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Abstract:

The invention relates to GLP-1 receptor agonist compounds with a modified
N-terminus. The compounds are of the formula Chem. 1: Y--Z--P, wherein P
represents a fragment of a GLP-1 receptor agonist peptide lacking the two
N-terminal amino acid residues; and Y--Z represents novel His-Ala
mimetics. Examples of GLP-1 receptor agonist compounds are derived from
human GLP-1 (7-37), exendin-4(1-39), or GLP-1 A (1-37). The invention
also relates to derivatives of these compounds, in particular compounds
with one or more albumin binding side chains capable of protracting the
duration of action in vivo of these compounds. The peptides and
derivatives of the invention have a good potency, a protracted
pharmacokinetic profile, are stable against degradation by gastro
intestinal enzymes, and/or have a high oral bioavailability. These
properties are of importance in the development of GLP-1 receptor agonist
compounds for subcutaneous, intravenous, and/or in particular oral
administration. The invention also relates to intermediate products for
use in the preparation of the GLP-1 receptor agonist compounds of the
invention.

3. A derivative of a peptide of claim 1, or a pharmaceutically acceptable
salt, amide, or ester thereof.

4. The derivative of claim 3 which has an albumin binding moiety attached
to a lysine residue of the peptide.

5. The derivative of claim 4, in which the albumin binding moiety
comprises a protracting moiety selected from Chem. 8, Chem. 9, and Chem.
10: HOOC--(CH2)x--CO--* Chem. 8
HOOC--C6H4--O--(CH2)y--CO--* Chem. 9
R18--C6H4--(CH2)z--CO--* Chem. 10 in which x is
an integer in the range of 6-18, y is an integer in the range of 3-17, z
is an integer in the range of 1-5, and R18 is a group having a molar
mass not higher than 150 Da.

6. The derivative of claim 5, wherein the albumin binding moiety further
comprises a linker selected from Chem. 11, Chem. 12, Chem. 13, and Chem.
14: *--NH--CH2--CH2--(O--CH2--CH2)k--O--(CH.sub-
.2)n--CO--* Chem. 11 *--NH--C(COOH)--(CH2)2--CO--* Chem.
12 *--N--C((CH2)2COOH)--CO--* Chem. 13
*--NC5H8--CO--* Chem. 14 wherein k is an integer in the range
of 1-5, and n is an integer in the range of 1-5.

16. A derivative of a peptide of claim 2, or a pharmaceutically
acceptable salt, amide, or ester thereof.

17. The derivative of claim 16 which has an albumin binding moiety
attached to a lysine residue of the peptide.

18. The derivative of claim 17, in which the albumin binding moiety
comprises a protracting moiety selected from Chem. 8, Chem. 9, and Chem.
10: HOOC--(CH2)x--CO--* Chem. 8
HOOC--C6H4--O--(CH2)y--CO--* Chem. 9
R18--C6H4--(CH2)z--CO--* Chem. 10 in which x is
an integer in the range of 6-18, y is an integer in the range of 3-17, z
is an integer in the range of 1-5, and R18 is a group having a molar
mass not higher than 150 Da.

19. The derivative of claim 18, wherein the albumin binding moiety
further comprises a linker selected from Chem. 11, Chem. 12, Chem. 13,
and Chem. 14:
*--NH--CH2--CH2--(O--CH2--CH2)k--O--(CH2).s-
ub.n--CO--* Chem. 11 *--NH--C(COOH)--(CH2)2--CO--* Chem. 12
*--N--C((CH2)2COOH)--CO--* Chem. 13 *--NC5H8--CO--*
Chem. 14 wherein k is an integer in the range of 1-5, and n is an
integer in the range of 1-5.

20. A pharmaceutical composition comprising a GLP-1 receptor agonist
peptide of claim 1 and a pharmaceutically acceptable carrier or diluent.

21. A method of treating diabetes in a subject in need of such treatment,
said method comprising administering to said subject an effective amount
of the pharmaceutical composition of claim 20.

22. A pharmaceutical composition comprising a GLP-1 receptor agonist
peptide of claim 2 and a pharmaceutically acceptable carrier or diluent.

23. A method of treating diabetes in a subject in need of such treatment,
said method comprising administering to said subject an effective amount
of the pharmaceutical composition of claim 22.

24. The derivative of claim 4, wherein the albumin binding moiety further
comprises a linker selected from the group consisting of: ##STR00048##

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to analogues and derivatives of GLP-1
receptor agonist peptides, and their pharmaceutical use. In the GLP-1
receptor agonist peptides of the invention, such as Glucagon-Like
Peptide-1 (GLP-1), exendins and analogues thereof, the two N-terminal
amino acids have been replaced by N-terminal mimetics.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

[0002] The Sequence Listing, entitled "SEQUENCE LISTING", is 1770 bytes,
was created on 1 Dec. 2010, and is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] WO 2004/067548 A2 relates to chemically modified metabolites of
regulatory peptides and methods of producing and using same.

[0004] Liraglutide, a GLP-1 derivative for once daily administration which
is marketed by Novo Nordisk A/S, is disclosed in Example 37 of WO
98/08871.

[0005] Semaglutide, a GLP-1 derivative for once weekly administration
which is under development by Novo Nordisk A/S, is disclosed in Example 4
of WO 06/097537.

[0007] Preferred compounds have the formula Chem. 1: Y--Z--P, wherein P
represents a fragment of a GLP-1 receptor agonist peptide lacking the
N-terminus; and Y--Z represents a group mimicking the N-terminus of the
peptide. The new N-terminal is preferably a His-Ala, a His-Gly, and/or a
His-Ser mimetic.

[0008] More in particular the invention relates to a GLP-1 receptor
agonist peptide having the formula Chem. 1: Y--Z--P, wherein P represents
a fragment of a GLP-1 receptor agonist peptide lacking the two N-terminal
amino acid residues; Z represents a group of the formula Chem. 2:

wherein q is 1-6, and R15 and R16 independently of each other and
independently for each value of q represent hydrogen, alkyl, carboxyl, or
hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceutically
acceptable salt, amide, or ester thereof.

[0010] The invention also relates to a derivative of this peptide, and a
pharmaceutically acceptable salt, amide, or ester thereof.

[0011] The invention also relates to the pharmaceutical use of these
compounds, preferably for the treatment and/or prevention of all forms of
diabetes and related diseases, such as eating disorders, cardiovascular
diseases, gastrointestinal diseases, diabetic complications, critical
illness, and/or polycystic ovary syndrome; and/or for improving lipid
parameters, improving β-cell function, and/or for delaying or
preventing diabetic disease progression.

[0012] Finally, the invention relates to intermediate products
corresponding to the new N-terminus, as well as to the peptide fragments,
i.e. before attachment of the new N-terminus, both relevant for the
preparation of the peptides of the invention.

[0013] The peptides and derivatives of the invention are biologically
active, preferably of a high potency. Also, or alternatively, they have a
protracted pharmacokinetic profile. Also, or alternatively, they are
stable against degradation by gastro intestinal enzymes. Also, or
alternatively, they have a high oral bioavailability. These properties
are of importance in the development of next generation GLP-1 compounds
for subcutaneous, intravenous, and/or in particular oral administration.

DESCRIPTION OF THE INVENTION

[0014] The invention relates to a GLP-1 receptor agonist peptide having
the formula Chem. 1: Y--Z--P, wherein P represents a fragment of a GLP-1
receptor agonist peptide lacking the two N-terminal amino acid residues;
Z represents a group of the formula Chem. 2:

wherein q is 1-6, and R15 and R16 independently of each other and
independently for each value of q represent hydrogen, alkyl, carboxyl, or
hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceutically
acceptable salt, amide, or ester thereof.

[0017] In a first aspect, R1 and R2 do not both represent hydrogen, and
the invention accordingly relates to a GLP-1 receptor agonist peptide
having the formula Chem. 1: Y--Z--P, wherein P represents a fragment of a
GLP-1 receptor agonist peptide lacking the two N-terminal amino acid
residues; Z represents a group of the formula Chem. 2:

wherein q is 1-6, and R15 and R16 independently of each other and
independently for each value of q represent hydrogen, alkyl, carboxyl, or
hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceutically
acceptable salt, amide, or ester thereof.

[0019] In a second aspect, R1 and R2 may both represent hydrogen, and
Q-NR--* is not attached to a nitrogen atom of Chem. 4.

[0020] In a third aspect, R1 and R2 may both represent hydrogen, and
Q-NR--* is attached to a carbon atom of Chem. 4.

[0021] The invention also relates to a derivative of each of these
peptides, and to pharmaceutically acceptable salts, amides, or esters
thereof.

[0022] The invention also relates to the pharmaceutical use of these
compounds, preferably for the treatment and/or prevention of all forms of
diabetes and related diseases, such as eating disorders, cardiovascular
diseases, gastrointestinal diseases, diabetic complications, critical
illness, and/or polycystic ovary syndrome; and/or for improving lipid
parameters, improving β-cell function, and/or for delaying or
preventing diabetic disease progression.

[0023] Finally, the invention relates to intermediate products
corresponding to the new N-terminus, as well as to the peptide fragments,
i.e. before attachment of the new N-terminus, both relevant for the
preparation of the peptides of the invention.

[0024] In what follows, Greek letters may be represented by their symbol
or the corresponding written name, for example: α=alpha;
β=beta; ε=epsilon; γ=gamma; ω=omega; etc. Also,
the Greek letter of μ may be represented by "u", e.g. in μl=ul, or
in μM=uM.

[0025] An asterisk (*) in a chemical formula designates i) a point of
attachment, ii) a radical, and/or iii) an unshared electron.

GLP-1 Receptor Agonist

[0026] The GLP-1 receptor agonist compounds of the invention may be
derived, or are derivable, from human GLP-1(7-37), exendin-4(1-39),
and/or GLP-1A(1-37). The amino acid sequences of these peptides may be
found in the UniProt Knowledgebase (UniProtKB)--SwissProt section
(www.uniprot.org) with the following accession numbers, sequence
identifiers, and sequence names: UNIPROT:P01275--8, GLUC_HUMAN,
Glucagon-like peptide 1(7-37); UNIPROT:P26349--3, EXE4_HELSU,
Exendin-4, or exenatide; and UNIPROT:042143--5, GLUC1_XENLA,
Glucagon-like peptide 1A; respectively.

[0027] The sequences of the corresponding fragments lacking the two
N-terminal amino acids, viz. GLP-1(9-37), exendin-4(3-39), and
GLP-1A(3-37), are included in the appended sequence listing as SEQ ID NO:
1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0028] Another example of a GLP-1 receptor agonist fragment from which the
compounds of the invention may be derived, or are derivable, is the
peptide designated exendin-3(3-39) which is the D3 analogue of SEQ ID NO:
2, i.e. identical to SEQ ID NO: 2 except for having aspartic acid (D,
Asp) at position 3, the first amino acid residue.

[0029] The sequence of the GLP-1 receptor may be found in the UniprotKB
database referred to above with the following accession number,
identifier, and name: UNIPROT:P43220, GLP1R_HUMAN, Glucagon-like peptide
1 receptor, GLP-1 receptor, GLP-1-R, or GLP-1R.

[0030] The term "GLP-1 receptor agonist" as used herein refers to a
compound which is an agonist of the human GLP-1 receptor, i.e. a compound
that stimulates the formation of cAMP in a medium containing the human
GLP-1 receptor. GLP-1 receptor agonism, or potency, is determined as
described below, in the section headed "Potency", see also Example 13
herein.

Amino Acids and Peptides

[0031] The term "peptide", as e.g. used in the context of the GLP-1
receptor agonist peptides of the invention, refers to a compound which
comprises a series of amino acids intereconnected by amide (or peptide)
bonds.

[0032] In a particular embodiment the peptide is to a large extent, or
predominantly, composed of amino acids interconnected by amide bonds
(e.g., at least 50%, 60%, 70%, 80%, or at least 90%, by molar mass). In
another particular embodiment the peptide consists of amino acids
interconnected by peptide bonds.

[0033] The peptides of the invention comprise at least five constituent
amino acids connected by peptide bonds. In particular embodiments the
peptide comprises at least 10, preferably at least 15, more preferably at
least 20, even more preferably at least 25, or most preferably at least
28 amino acids.

[0034] In particular embodiments, the peptide is composed of at least five
constituent amino acids, preferably composed of at least 10, at least 15,
at least 20, at least 25, or most preferably composed of at least 28
amino acids.

[0037] In a still further particular embodiment the peptide consists of
amino acids interconnected by peptide bonds.

[0038] Amino acids are molecules containing an amine group and a
carboxylic acid group, and, optionally, one or more additional groups,
often referred to as a side chain.

[0039] The term "amino acid" includes proteogenic amino acids (encoded by
the genetic code, including natural amino acids, and standard amino
acids), as well as non-proteogenic (not found in proteins, and/or not
coded for in the standard genetic code), and synthetic amino acids. Thus,
the amino acids may be selected from the group of proteinogenic amino
acids, non-proteinogenic amino acids, and/or synthetic amino acids.

[0040] Non-limiting examples of amino acids which are not encoded by the
genetic code are gamma-carboxyglutamate, ornithine, and phosphoserine.
Non-limiting examples of synthetic amino acids are the D-isomers of the
amino acids such as D-alanine and D-leucine, Aib (α-aminoisobutyric
acid), β-alanine, and des-amino-histidine (desH, alternative name
imidazopropionic acid, abbreviated Imp).

[0041] In what follows, all amino acids for which the optical isomer is
not stated is to be understood to mean the L-isomer (unless otherwise
specified).

[0042] A "GLP-1 receptor agonist peptide" is a peptide as defined above,
and also a GLP-1 receptor agonist as defined above.

[0043] The peptides of the invention are GLP-1 receptor agonist peptides.

[0044] Additional examples of GLP-1 receptor agonist peptides are the
following known compounds: Human GLP-1(7-37), exendin-4(1-39),
exendin-3(1-39), and GLP-1A(1-37).

[0045] In a particular embodiment, the GLP-1 receptor agonist compound of
the invention may be derived, or is derivable, from any one or more of
these known GLP-1 receptor agonist peptides.

[0046] The term "fragment" as it refers to a GLP-1 receptor agonist
peptide means a peptide which is shorter than the peptide referred to.

[0047] In a particular embodiment corresponding to the definition of group
P in formula I, the fragment lacks the two N-terminal amino acids as
compared to the corresponding full-length peptide being a GLP-1 receptor
agonist.

[0048] In another particular embodiment this particular fragment is not in
itself a GLP-1 receptor agonist, due to a i) substantial, ii) preferably
almost complete, or iii) more preferably for all practical purposes
complete, loss of biological activity (i.e., GLP-1 receptor agonism).

[0049] Particular examples of P (fragments of a GLP-1 receptor agonist
peptide lacking the two N-terminal amino acid residues) are the
following: GLP-1(9-37), exendin-4(3-39), and GLP-1A(3-37), which are
included in the appended sequence listing as SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3, respectively. Another example of P is exendin-3(3-39)
which is variant D3 of SEQ ID NO: 2.

[0050] In the sequence listing, the first amino acid residue of the
fragment of GLP-1(9-37) (SEQ ID NO: 1), which is glutamic acid, is
assigned no. 1. However, in what follows--according to established
practice in the art--this glutamic acid residue is referred to as no. 9,
and subsequent amino acid residues are numbered accordingly, ending with
glycine no. 37.

[0051] Likewise, in the sequence listing, the first amino acid residue of
exendin-4(3-39) (SEQ ID NO: 2), which is also glutamic acid, is assigned
no. 1. However, in what follows--according to established practice in the
art--this glutamic acid residue is referred to as no. 3, and subsequent
amino acid residues are numbered accordingly, ending with serine as no.
39.

[0052] Likewise, in the sequence listing, the first amino acid residue of
GLP-1A(3-37) (SEQ ID NO: 3), which is aspartic acid, is assigned no. 1.
However, in what follows--according to established practice in the
art--this aspartic acid residue is referred to as no. 3, and subsequent
amino acid residues are numbered accordingly, ending with serine as no.
37.

[0053] Therefore, generally, any reference herein to an amino acid residue
number or a position number in the context of the peptides of SEQ ID NO:
1, 2, or 3 or analogues thereof is to the sequence starting with Glu at
position 9, Glu at position 3, or Asp at position 3, respectively; and
ending with Gly at position 37, Ser at position 39, or Ser at pos. 37,
respectively.

[0055] An "analogue" as used herein in the context of SEQ ID NO: 1, 2, or
3 refers to a peptide, or a compound, which is a variant of any one or
more of SEQ ID NO: 1, 2, or 3.

[0056] In a particular embodiment, the analogue of SEQ ID NO: 1 refers to
a modification of SEQ ID NO: 1 in which a number of amino acid residues
have been exchanged as compared to SEQ ID NO: 1. These exchanges, or
modifications, may represent, independently, one or more amino acid
substitutions, additions, and/or deletions. Additions at the N-terminus
are, however, preferably excluded. Analogues of SEQ ID NO: 2, and SEQ ID
NO: 3 are defined similarly, by analogy to the definition of analogues of
SEQ ID NO: 1.

[0057] Analogues may be described by reference to a reference sequence,
the number of the amino acid residue in the reference sequence
corresponding to the one which is modified, i.e., its position, and to
the actual modification.

[0059] The following are non-limiting, illustrative examples of suitable
analogue nomenclature, as used herein:

[0060] N9-[-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propion-
yl}-[Lys18,Glu22,Gln34]GLP-1(9-37) peptide is a GLP-1
receptor agonist peptide of the invention derivable from GLP-1(9-37) (SEQ
ID NO: 1), i.e. P in Chem. 1 is an analogue of SEQ ID NO: 1, viz. the
analogue in which the serine at position 18 has been substituted with
lysine, the glycine at position 22 has been substituted with glutamic
acid, and the lysine at position 34 has been substituted with glutamine;

[0061] N9-{2-[2-(1H-Imidazol-4-yl)propylcarbamoyl]-2-methyl-propionyl-
}-[Glu30,Lys36]GLP-1(9-37)Glu38-peptide is a GLP-1 receptor
agonist peptide of the invention derivable from GLP-1(9-37) (SEQ ID NO:
1), i.e. P in Chem. 1 is an analogue of SEQ ID NO: 1, viz. the analogue
in which the alanine at position 30 has been substituted with glutamic
acid, the arginine at position 36 has been substituted with lysine, and a
glutamic acid has been added at the C-terminus, viz. at position 38; and

[0062] N3-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propiony-
l}-[Arg17,Arg20,Arg33,Lys38]GLP-1A(3-37)-peptide is a
GLP-1 receptor agonist peptide of the invention derivable from
GLP-1A(3-37) (SEQ ID NO: 3), i.e. P in Chem. 1 is an analogue of SEQ ID
NO: 3, viz. the analogue in which the lysines at position 17, 20, and 33
have been substituted with arginine, and a lysine has been added at the
C-terminus, viz. at position 38.

[0064] As another example, a GLP-1 receptor agonist peptide of the
invention which "comprises at least one of the following substitutions as
compared to GLP-1(9-37) (SEQ ID NO: 1): 18K; 22E; 30E; 31H; 34Q,R; 36K;
37K; and/or 38E" refers to a GLP-1 receptor agonist peptide in which P of
Chem. 1 is considered an analogue of SEQ ID NO: 1, which analogue has a
lysine at position 18, a glutamic acid at position 22, a glutamic acid at
position 30, a histidine at position 31, a glutamine at position 34, a
lysine at position 36, a lysine at position 37, and/or a glutamic acid at
position 38, and which analogue may comprise further modifications as
compared to SEQ ID NO: 1.

[0065] As is apparent from the above examples, amino acid residues may be
identified by their full name, their one-letter code, and/or their
three-letter code. These three ways are fully equivalent.

[0066] The expressions "a position equivalent to" or "corresponding
position" may be used to characterise the site of modification in a
modified GLP-1 receptor agonist peptide sequence by reference to any one
or more of SEQ ID NO: 1, 2, or 3. Equivalent or corresponding positions,
as well as the number of modifications, are easily deduced, e.g. by
simple handwriting and eyeballing; and/or a standard protein or peptide
alignment program may be used, such as "align" which is a
Needleman-Wunsch alignment. The algorithm is described in Needleman, S.
B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48: 443-453,
and the align program by Myers and W. Miller in "Optimal Alignments in
Linear Space" CABIOS (computer applications in the biosciences) (1988)
4:11-17. For the alignment, the default scoring matrix BLOSUM50 and the
default identity matrix may be used, and the penalty for the first
residue in a gap may be set at -12, or preferably at -10, and the
penalties for additional residues in a gap at -2, or preferably at -0.5.

[0067] This algorithm may also suitably be used for determining the degree
of identity of the P-group of a GLP-1 receptor agonist peptide of the
invention to each of SEQ ID NO: 1, 2, and 3, e.g. with a view to
determining which of these three sequences has the highest percentage of
identity to the P-group in question, and thus for determining the number
of amino acid residues that have been exchanged as compared to the
closest related sequence of SEQ ID NOs: 1-3 (the one with the highest
percentage of identity). If the percentages of identity of a given P
group of a GLP-1 receptor agonist of the invention to SEQ ID NO: 1, 2,
and 3, respectively, should happen to be the same, any of those having
the same highest percentage of identity may be used for the
determination. An example of such alignment is inserted hereinbelow, in
which sequence no. 1 is SEQ ID NO: 1, and sequence no. 2 is SEQ ID NO: 3:

# 1: GLP--1(9-37)

# 2: GLP-1A(3-37)

# Matrix: EBLOSUM62

# Gap_penalty: 10.0

# Extend_penalty: 0.5

# Length: 35

# Identity: 18/35 (51.4%)

# Similarity: 22/35 (62.9%)

# Gaps: 6/35 (17.1%)

# Score: 92.0

##STR00013##

[0069] The above alignment is just for illustration, as typically an
analogue of one of SEQ ID NOs: 1, 2, or 3 will be compared with either of
these reference sequences.

[0070] In case of analogues comprising non-natural amino acids such as
Imp, and/or Aib being included in the sequence, these may, for alignment
purposes, be replaced with X. If desired, X can later be manually
corrected.

Derivatives

[0071] The terms "derivative" as used herein in the context of the GLP-1
receptor agonist peptides of the invention means a chemically modified
peptide or analogue, in which one or more substituents have been
covalently attached to the peptide. The substituent(s) may also be
referred to as side chain(s). In a particular embodiment, the derivative
of the invention has one side chain. In another particular embodiment it
has two side chains. For the purpose of this definition, the group Y--Z--
of formula I is preferably not considered a substituent/side chain.

[0072] In particular embodiments, the side chain has at least 10 carbon
atoms, or at least 15, 20, 25, 30, 35, 40, or at least 43 carbon atoms.
In further particular embodiments, the side chain may further include at
least 5 hetero atoms, in particular O and N, for example at least 7, 9,
10, 12, 15, 17, or at least 20 hetero atoms, such as at least 1, 2, 3, or
4 N-atoms, and/or at least 3, 4, 6, 9, 12, 13, or 15 O-atoms.

[0073] Non-limiting examples of GLP-1 receptor agonist derivatives include
heterologous fusion proteins or conjugates of the GLP-1 receptor agonist
peptides of the invention, with e.g. the Fc portion of an immunoglobulin
such as IgG, with human albumin, with antibodies such as a glucagon
binding antibody heavy chain variable region, or with fragments or
analogues of any of these (see, e.g., US 2007/0161087, WO 2005/058958,
and WO 2007/124463 A2). Additional examples include PEGylated peptides
(see, e.g., WO 2005/058954, WO 2004/093823, and WO 2006/124529), as well
as acylated peptides (see, e.g., WO 98/08871, WO2005/027978, WO
2006/097537, and WO 2009/030771).

[0074] In a preferred embodiment, the side chain is capable of forming
non-covalent aggregates with albumin, thereby promoting the circulation
of the derivative with the blood stream, and also having the effect of
protracting the time of action of the derivative, due to the fact that
the aggregate of the derivative and albumin is only slowly disintegrated
to release the active pharmaceutical ingredient. Thus, a preferred
substituent, or side chain, as a whole may be referred to as an albumin
binding moiety.

[0075] In another particular embodiment, the albumin binding moiety
comprises a portion which is particularly relevant for the albumin
binding and thereby the protraction, which portion may accordingly be
referred to as a protracting moiety. The protracting moiety may be at, or
near, the opposite end of the albumin binding moiety, relative to its
point of attachment to the peptide.

[0076] In a still further particular embodiment, the albumin binding
moiety comprises a portion in-between the protracting moiety and the
point of attachment to the peptide, which portion may be referred to as a
linker, linker moiety, spacer, or the like. The presence of a linker is
optional; hence if no linker is present the albumin binding moiety may be
identical to the protracting moiety.

[0078] The albumin binding moiety, the protracting moiety, or the linker
may be covalently attached to a lysine residue of the GLP-1 receptor
agonist peptide by conjugation chemistry such as by alkylation,
acylation, ester formation, or amide formation; or to a cysteine residue,
such as by maleimide or haloacetamide (such as bromo-/fluoro-/iodo-)
coupling.

[0079] In a preferred embodiment, an active ester of the albumin binding
moiety and/or the protracting moiety, optionally with a linker, is
covalently linked to an amino group of a lysine residue, preferably the
epsilon amino group thereof, under formation of an amide bond (this
process being referred to as acylation).

[0080] Unless otherwise stated, when reference is made to an acylation of
a lysine residue, it is understood to be to the epsilon-amino group
thereof.

[0081] In one embodiment, the invention relates to a derivative of a GLP-1
receptor agonist peptide which comprises, preferably has, an albumin
binding moiety attached to one or more of 18K, 26K, 36K, and/or 37K,
wherein reference may be had to the sequence of GLP-1(9-37) (SEQ ID NO:
1). As explained above, each residue number refers to the corresponding
position in GLP-1(9-37) (SEQ ID NO: 1). Furthermore, as also explained
above, ordinary script may be used instead of superscript to designate
the position number. E.g., "K18" is fully equivalent to "18K".

[0082] Corresponding position numbers are preferably identified by
handwriting and eyeballing, or by using a suitable alignment program, as
explained above.

[0083] For the present purposes, the terms "albumin binding moiety",
"protracting moiety", and "linker" include the un-reacted as well as the
reacted forms of these molecules. Whether or not one or the other form is
meant is clear from the context in which the term is used.

[0084] In one aspect the albumin binding moiety comprises, or consists of,
a protracting moiety selected from

HOOC--(CH2)x--CO--* Chem. 8

HOOC--C6H4--O--(CH2)y--CO--* Chem. 9

R18--C6H4--(CH2)z--CO--* Chem. 10

[0085] in which x is an integer in the range of 6-18, y is an integer in
the range of 3-17, z is an integer in the range of 1-5, and R18 is a
group having a molar mass not higher than 150 Da.

[0086] In one embodiment, *--(CH2)x--* refers to straight or
branched, preferably straight, alkylene in which x is an integer in the
range of 6-18.

[0087] In another embodiment, *--(CH2)y--* refers to straight or
branched, preferably straight, alkylene in which y is an integer in the
range of 3-17.

[0088] In a third embodiment, *--(CH2)z--* refers to straight or
branched, preferably straight, alkylene in which z is an integer in the
range of 1-5.

[0089] The molar mass (M) of a chemical substance (such as the group
R1) is the mass of one mole of the substance. The molar mass is
quoted in dalton, symbol Da, with the definition 1 Da=1 g/mol.

[0090] Molar mass may be calculated from standard atomic weights, and is
often listed in chemical catalogues. The molar mass of a compound is
given by the sum of the standard atomic weights of the atoms which form
the compound multiplied by the molar mass constant, Mu which equals
1 g/mol. As an example, the molecular mass of tert. butyl
(C4H9) is
M(C4H9)═([4×12.01]+[9×1.008])×1 g/mol=57
Da.

[0091] Standard atomic weights are published by the International Union of
Pure and Applied Chemistry (IUPAC), and also reprinted in a wide variety
of textbooks, commercial catalogues, wallcharts etc.

[0092] For the attachment to the GLP-1 receptor agonist peptide, the acid
group of the fatty acid, or one of the acid groups of the fatty diacid,
forms an amide bond with the epsilon amino group of a lysine residue in
the GLP-1 receptor agonist peptide.

[0093] The term "fatty acid" refers to aliphatic monocarboxylic acids
having from 4 to 28 carbon atoms, it is preferably unbranched, and/or
even numbered, and it may be saturated or unsaturated.

[0094] The term "fatty diacid" refers to fatty acids as defined above but
with an additional carboxylic acid group in the omega position. Thus,
fatty diacids are dicarboxylic acids.

[0095] The nomenclature is as is usual in the art, for example *--COOH, as
well as HOOC--*, refers to carboxy; *--C6H4--* to phenylene;
*--CO--*, as well as *--OC--*, to carbonyl (O═C<**); and
C6H5--O--* to phenoxy.

[0096] In particular embodiments, the aromatics, such as the phenoxy and
the phenylene radicals, may be, independently, ortho, meta, or para.

[0097] In a preferred embodiment the linker moiety, if present, has from 5
to 30 C-atoms. In additional preferred embodiments, the linker moiety, if
present, has from 4 to 20 hetero atoms. H-atoms are not hetero atoms.

[0098] In another embodiment, the linker comprises at least one OEG
molecule, at least one glutamic acid residue, and/or at least one
piperidine molecule, optionally substituted, or rather the corresponding
radicals (OEG designates 8-amino-3,6-dioxaoctanic acid, i.e. this
di-radical:
*--NH--(CH2)2--O--(CH2)2--O--CH2--CO--*).

[0099] The amino acid glutamic acid comprises two carboxylic acid groups.
Its gamma-carboxy group is preferably used for forming an amide bond with
the epsilon-amino group of lysine, or with an amino group of an OEG
molecule, if present, or with the amino group of another Glu residue, if
present. The amino group of Glu in turn forms an amide bond with the
carboxy group of the protracting moiety, or with the carboxy group of an
OEG molecule, if present, or with the gamma-carboxy group of another Glu,
if present. This way of inclusion of Glu is occasionally briefly referred
to as "gamma-Glu".

[0100] The derivatives of the invention may exist in different
stereoisomeric forms having the same molecular formula and sequence of
bonded atoms, but differing only in the three-dimensional orientation of
their atoms in space. The stereoisomerism of the exemplified derivatives
of the invention is indicated in the experimental section, in the names
as well as the structures, using standard nomenclature. Unless otherwise
stated the invention relates to all stereoisomeric forms of the claimed
derivative.

[0101] The concentration in plasma of the GLP-1 receptor agonist peptides
and derivatives of the invention may be determined using any suitable
method. For example, LC-MS (Liquid Chromatography Mass Spectroscopy) may
be used, or immunoassays such as RIA (Radio Immuno Assay), ELISA
(Enzyme-Linked Immuno Sorbent Assay), and LOCI (Luminescence Oxygen
Channeling Immunoasssay). General protocols for suitable RIA and ELISA
assays are found in, e.g., WO09/030,738 on p. 116-118. A preferred assay
is the LOCI assay in which the plasma concentrations of the compounds are
determined using a Luminescence Oxygen Channeling Immunoasssay (LOCI),
generally as described for the determination of insulin by Poulsen and
Jensen in Journal of Biomolecular Screening 2007, vol. 12, p. 240-247.
The donor beads are coated with streptavidin, while acceptor beads are
conjugated with a monoclonal antibody recognising a mid-/C-terminal
epitope of the peptide. Another monoclonal antibody, specific for the
N-terminus, is biotinylated. The three reactants are combined with the
analyte and form a two-sited immuno-complex. Illumination of the complex
releases singlet oxygen atoms from the donor beads, which are channeled
into the acceptor beads and trigger chemiluminescence which is measured
in an Envision plate reader. The amount of light is proportional to the
concentration of the compound.

Pharmaceutically Acceptable Salt, Amide, or Ester

[0102] The GLP-1 receptor agonist peptides, derivatives, and intermediate
products of the invention may be in the form of a pharmaceutically
acceptable salt, amide, or ester.

[0103] Salts are e.g. formed by a chemical reaction between a base and an
acid, e.g.: NH3+H2SO4→(NH4)2SO4.

[0104] The salt may be a basic salt, an acid salt, or it may be neither
nor (i.e. a neutral salt). Basic salts produce hydroxide ions and acid
salts hydronium ions in water.

[0105] The salts of the peptides and derivatives of the invention may be
formed with added cations or anions that react with anionic or cationic
groups, respectively. These groups may be situated in the peptide moiety,
and/or in the side chain of the compounds of the invention.

[0106] Non-limiting examples of anionic groups of the compounds of the
invention include free carboxylic groups in the side chain, if any, as
well as in the peptide moiety. The peptide moiety often includes a free
carboxylic acid group at the C-terminus, and it may also include free
carboxylic groups at internal acid amino acid residues such as Asp and
Glu.

[0107] Non-limiting examples of cationic groups in the peptide moiety
include the free amino group at the N-terminus, if present, as well as
any free amino group of internal basic amino acid residues such as His,
Arg, and Lys.

[0108] The ester of the peptides and derivatives of the invention may,
e.g., be formed by the reaction of a free carboxylic acid group with an
alcohol or a phenol, which leads to replacement of at least one hydroxyl
group by an alkoxy or aryloxy group

[0109] The ester formation may involve the free carboxylic group at the
C-terminus of the peptide, and/or any free carboxylic group in the side
chain.

[0110] The amide of the peptides and derivatives of the invention may,
e.g., be formed by the reaction of a free carboxylic acid group with an
amine or a substituted amine, or by reaction of a free or substituted
amino group with a carboxylic acid.

[0111] The amide formation may involve the free carboxylic group at the
C-terminus of the peptide, any free carboxylic group in the side chain,
the free amino group at the N-terminus of the peptide, and/or any free or
substituted amino group of the peptide in the peptide and/or the side
chain.

[0112] In a particular embodiment, the peptide or derivative is in the
form of a pharmaceutically acceptable salt. In another particular
embodiment, the peptide or derivative is in the form of a
pharmaceutically acceptable amide, preferably with an amide group at the
C-terminus of the peptide. In a still further particular embodiment, the
peptide or derivative is in the form a pharmaceutically acceptable ester.

Intermediate Compounds

[0113] The invention also relates to an intermediate product of the
formula Chem. 50 or Chem. 51:

##STR00014##

wherein Q, R, R1, and R2 are as defined for the GLP-1 receptor agonist
peptide of the invention, having the formula Chem. 1, and each of
PG1 and PG2 represents a protection group.

[0115] Non-limiting examples of PG2 groups are --OH, or groups
functionalised as an activated ester, for example, without limitation,
OPfp, OPnp, and OSuc.

[0116] Other suitable activated esters may be selected, e.g., according to
the teaching of M. Bodanszky, "Principles of Peptide Synthesis", 2nd ed.,
Springer Verlag, 1993.

Functional Properties

[0117] In a first functional aspect, the GLP-1 receptor agonist peptides
and/or derivatives of the invention have a good potency. Also, or
alternatively, in a second functional aspect, they have a protracted
pharmacokinetic profile. Also, or alternatively, in a third functional
aspect, they are stable against degradation by gastro intestinal enzymes.
Also, or alternatively, in a fourth functional aspect, they have a high
oral bioavailability.

[0118] Biological Activity (Potency)

[0119] According to the first functional aspect, the GLP-1 receptor
agonist derivatives are biologically active, or have a good potency.

[0120] Surprisingly, they have an improved potency as compared to the
comparative compound of Example 11 (Chem. 40) herein, which is based on
one of the most potent compounds of the prior art, viz. compound 215 of
WO2004/067548.

[0121] Also, or additionally, the derivatives of the invention have a high
binding affinity to the GLP-1 receptor at low albumin concentration
(0.005%), i.e. a low IC50 value, which is discussed further below
under the heading of receptor binding.

[0122] As regards potency, the term half maximal effective concentration
(EC50) generally refers to the concentration which induces a
response halfway between the baseline and maximum, by reference to the
dose response curve. EC50 is used as a measure of the potency of a
compound and represents the concentration where 50% of its maximal effect
is observed.

[0123] The in vitro potency of the derivatives of the invention may be
determined as described hereinbelow, and the EC50 of the derivative
in question determined. The lower the EC50, the better the potency.

[0124] In a particular embodiment, the derivatives of the invention are at
least 3 times more potent than Chem. 40; preferably at least 4 times more
potent; even more preferably at least 5 times more potent; or most
preferably at least 6 times more potent than Chem. 40.

[0125] In another particular embodiment, the derivatives of the invention
are at least 7 times more potent than Chem. 40; preferably at least 8
times more potent; even more preferably at least 9 times more potent; or
most preferably at least 10 times more potent than Chem. 40.

[0126] In another particular embodiment, the derivatives of the invention
are at least 20 times more potent than Chem. 40; preferably at least 50
times more potent; even more preferably at least 100 times more potent;
still more preferably at least 200 times more potent; or most preferably
at least 400 times more potent than Chem. 40.

[0127] Potency is preferably determined as described below, and it is
noted that a, e.g., three times more potent compound has an EC50
which is three times lower.

[0128] In a particular embodiment, potency and/or activity refers to in
vitro potency, i.e. performance in a functional GLP-1 receptor assay,
more in particular to the capability of stimulating cAMP formation in a
cell line expressing the cloned human GLP-1 receptor.

[0129] The stimulation of the formation of cAMP in a medium containing the
human GLP-1 receptor may preferably be determined using a stable
transfected cell-line such as BHK467-12A (tk-ts13), and/or using for the
determination of cAMP a functional receptor assay, e.g. based on
competition between endogenously formed cAMP and exogenously added
biotin-labelled cAMP, in which assay cAMP is more preferably captured
using a specific antibody, and/or wherein an even more preferred assay is
the AlphaScreen cAMP Assay, most preferably the one described in Example
13.

[0132] In another particular embodiment the derivatives of the invention
are potent in vivo, which may be determined as is known in the art in any
suitable animal model, as well as in clinical trials. The diabetic db/db
mouse is one example of a suitable animal model, and the blood glucose
lowering effect may be determined in such mice in vivo, e.g. as described
in Example 43 of WO09/030,738.

Protraction--Receptor Binding--High and Low Albumin

[0133] According to the second functional aspect, the derivatives of the
invention are protracted.

[0134] A suitable assay for determining receptor binding of the peptides
and derivatives of the invention at high and low albumin concentration is
disclosed in Example 14 herein. Generally, the binding to the GLP-1
receptor at low albumin concentration should be as good as possible,
corresponding to a low IC50 value.

[0135] The IC50 value at high albumin concentration is a measure of
the influence of albumin on the binding of the compound to the GLP-1
receptor. As is known, the peptides GLP-1 receptor agonist peptide
derivatives of the invention also bind to albumin. This is a generally
desirable effect, which extends their lifetime in plasma. Therefore, the
IC50 value at high albumin will generally be higher than the
IC50 value at low albumin, corresponding to a reduced binding to the
GLP-1 receptor, caused by albumin binding competing with the binding to
the GLP-1 receptor.

[0136] A high ratio (IC50 value (high albumin)/IC50 value (low
albumin)) may therefore be taken as an indication that the derivative in
question binds well to albumin (may have a long half-life), and also per
se binds well to the GLP-1 receptor (the IC50 value (high albumin)
is high, and the IC50 value (low albumin) is low). On the other
hand, albumin binding may not always be desirable, or the binding to
albumin may become too strong. Therefore, the desirable ranges for
IC50 (low albumin), IC50 (high albumin)/, and the ratio
high/low may vary from compound to compound, depending on the intended
use and the circumstances surrounding such use, and on other compound
properties of potential interest.

[0137] In a particular embodiment, the peptides and derivatives of the
invention have a high binding affinity to the GLP-1 receptor at low
albumin concentration (0.005%), i.e. a low IC50 value.

[0139] According to the third functional aspect, the GLP-1 receptor
agonist peptides and/or derivatives of the invention are stable, or
stabilised, against degradation by one or more gastro intestinal enzymes.

[0140] Gastro intestinal enzymes include, without limitation, exo and endo
peptidases, such as pepsin, trypsin, chymotrypsin, elastases, and
carboxypeptidases. The stability may be tested against these gastro
intestinal enzymes in the form of purified enzymes, or in the form of
extracts from the gastrointestinal system.

[0141] In a particular embodiment, the derivative of the invention has an
in vitro half-life (T1/2), in an extract of rat small intestines,
divided by the corresponding half-life (T1/2) of GLP-1(7-37), of
above 1.0, preferably above 2.0, more preferably above 3.0, even more
preferably above 4.0, or most preferably above 5.0. In other words, a
ratio (SI) may be defined for each derivative, viz. as the in vitro
half-life (T1/2) of the derivative in question, in an extract of rat
small intestines, divided by the corresponding half-life (T1/2) of
GLP-1(7-37).

[0142] A suitable assay for determining in vitro half-life in an extract
of rat small intestines is disclosed in Example 57 of a PCT application
entitled "Double-acylated GLP-1 derivatives" filed with the EPO as
receiving office, by Novo Nordisk A/S, on the same date as the present
application.

Protraction--Half Life In Vivo in Rats

[0143] According to the second functional aspect, the derivatives of the
invention are protracted. In a particular embodiment, protraction may be
determined as half-life (T1/2) in vivo in rats after i.v.
administration. In additional embodiments, the half-life is at least 4
hours, preferably at least 5 hours, even more preferably at least 6
hours, or most preferably at least 8 hours.

[0144] A suitable assay for determining half-life in vivo in rats after
i.v. administration is disclosed in Example 58 of a PCT application
entitled "Double-acylated GLP-1 derivatives" filed with the EPO as
receiving office, by Novo Nordisk A/S, on the same date as the present
application.

Protraction--Half Life In Vivo in Minipigs

[0145] According to the second functional aspect, the derivatives of the
invention are protracted. In a particular embodiment protraction may be
determined as half-life (T1/2) in vivo in minipigs after i.v.
administration. In additional embodiments, the half-life is at least 12
hours, preferably at least 24 hours, more preferably at least 36 hours,
even more preferably at least 48 hours, or most preferably at least 60
hours.

[0146] A suitable assay for determining half-life in vivo in minipigs
after i.v. administration is disclosed in Example 16 herein.

Oral Bioavailability

[0147] According to the fourth functional aspect, the derivatives of the
invention have a high oral bioavailability.

[0148] The oral bioavailability of commercial GLP-1 receptor agonist
peptide derivatives is very low. The oral bioavailability of such
derivatives under development for i.v. or s.c. administration is also
low.

[0149] Accordingly, there is a need in the art for derivatives of an
improved oral bioavailability. Such derivatives could be suitable
candidates for oral administration, as long as their potency is generally
satisfactory, and/or as long as their half-life is also generally
satisfactory.

[0150] The present inventors identified a novel class of GLP-1 receptor
agonist peptide derivatives, which have a high oral bioavailability, and
at the same time a satisfactory potency, and/or half-life.

[0151] Also, or alternatively, these derivatives have a high oral
bioavailability, and at the same time a high binding affinity (i.e. a low
IC50 value) to the GLP-1 receptor at a low concentration of albumin.

[0152] These features are of importance with a view to obtaining a low
daily oral dose of the active pharmaceutical ingredient, which is
desirable for various reasons, including, e.g., economy of production,
likelihood of potential safety issues, as well as administration comfort
issues, and environmental concerns.

[0153] Generally, the term bioavailability of a GLP-1 receptor agonist
compound of the invention refers to the fraction of an administered dose
of the compound that reaches the systemic circulation unchanged. By
definition, when a medication is administered intravenously, its
bioavailability is 100%. However, when a medication is administered via
other routes (such as orally), its bioavailability decreases (due to
incomplete absorption and first-pass metabolism). Knowledge about
bioavailability is essential when calculating dosages for non-intravenous
routes of administration.

[0154] Absolute oral bioavailability compares the bioavailability
(estimated as the area under the curve, or AUC) of the active drug in
systemic circulation following oral administration, with the
bioavailability of the same drug following intravenous administration. It
is the fraction of the drug absorbed through non-intravenous
administration compared with the corresponding intravenous administration
of the same drug. The comparison must be dose normalised if different
doses are used; consequently, each AUC is corrected by dividing the
corresponding dose administered.

[0155] A plasma drug concentration vs time plot is made after both oral
and intravenous administration. The absolute bioavailability (F) is the
dose-corrected AUC-oral divided by AUC-intravenous.

[0156] The GLP-1 receptor agonist compounds of the invention have an
absolute oral bioavailability which is higher than that of a)
liraglutide, and/or b) semaglutide; preferably at least 10% higher, more
preferably at least 20% higher, even more preferably at least 30% higher,
or most preferably at least 40% higher. Before testing oral
bioavailability the GLP-1 receptor agonist compounds of the invention may
suitably be formulated as is known in the art of oral formulations of
insulinotropic compounds, e.g. using any one or more of the formulations
described in WO 2008/145728.

[0157] A test has been developed, described in Example 15, which was found
to be a very good prediction of oral bioavailability. According to this
test, after direct injection of a GLP-1 derivative into the intestinal
lumen of rats, the concentration (exposure) thereof in plasma is
determined, and the ratio of plasma concentration (pmol/l) divided by the
concentration of the dosing solution (umol/l) is calculated for t=30 min.
This ratio is a measure of intestinal bioavailability, and it has shown
to correlate nicely with actual oral bioavailability data.

[0158] Additional particular embodiments of the derivatives of the
invention are described in the section headed "particular embodiments"
before the experimental section.

Production Processes

[0159] The production of peptides like for example GLP-1(7-37) and
analogues thereof is well known in the art.

[0160] The fragment P of the peptides of formula I of the invention may
for instance be produced by classical peptide synthesis, e.g., solid
phase peptide synthesis using t-Boc or Fmoc chemistry or other well
established techniques, see, e.g., Greene and Wuts, "Protective Groups in
Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza Dorwald,
"Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH, 2000, and
"Fmoc Solid Phase Peptide Synthesis", Edited by W. C. Chan and P. D.
White, Oxford University Press, 2000.

[0161] Also, or alternatively, it may be produced by recombinant methods,
viz. by culturing a host cell containing a DNA sequence encoding the
fragment and capable of expressing the peptide in a suitable nutrient
medium under conditions permitting the expression of the peptide.
Non-limiting examples of host cells suitable for expression of these
peptides are: Escherichia coli, Saccharomyces cerevisiae, as well as
mammalian BHK or CHO cell lines.

[0162] The complete GLP-1 receptor agonist peptides of the invention
incorporating, viz. adding Y--Z to P of formula I, may e.g. be produced
as described in the experimental part. Or see Hodgson et al: "The
synthesis of peptides and proteins containing non-natural amino acids",
Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430; and in WO
2009/083549 A1 entitled "Semi-recombinant preparation of GLP-1
analogues".

[0163] Derivatives of the invention may be prepared as is known in the
art, and specific examples of methods of preparing a number of
derivatives of the invention are included in the experimental part
herein.

Pharmaceutical Compositions

[0164] Pharmaceutical compositions comprising a peptide or a derivative of
the invention; or a pharmaceutically acceptable salt, amide, or ester
thereof, and a pharmaceutically acceptable excipient may be prepared as
is known in the art.

[0165] The term "excipient" broadly refers to any component other than the
active therapeutic ingredient(s). The excipient may be an inert
substance, an inactive substance, and/or a not medicinally active
substance.

[0166] The excipient may serve various purposes, e.g. as a carrier,
vehicle, diluent, tablet aid, and/or to improve administration, and/or
absorption of the active substance.

[0167] The formulation of pharmaceutically active ingredients with various
excipients is known in the art, see e.g. Remington: The Science and
Practice of Pharmacy (e.g. 19th edition (1995), and any later
editions).

[0169] Examples of formulations include liquid formulations, i.e. aqueous
formulations comprising water. A liquid formulation may be a solution, or
a suspension. An aqueous formulation typically comprises at least 50% w/w
water, or at least 60%, 70%, 80%, or even at least 90% w/w of water.

[0170] Alternatively, a pharmaceutical composition may be a solid
formulation, e.g. a freeze-dried or spray-dried composition, which may be
used as is, or whereto the physician or the patient adds solvents, and/or
diluents prior to use.

[0171] The pH in an aqueous formulation may be anything between pH 3 and
pH 10, for example from about 7.0 to about 9.5; or from about 3.0 to
about 7.0.

[0172] A pharmaceutical composition may comprise a buffer. The buffer may
e.g. be selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine,
sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium
phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic
acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid,
and mixtures thereof. A pharmaceutical composition may comprise a
preservative. The preservative may e.g. be selected from the group
consisting of phenol, o-cresol, m-cresol, p-cresol, methyl
p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl
p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and
thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium
dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium
chloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol), and mixtures
thereof. The preservative may be present in a concentration from 0.1
mg/ml to 20 mg/ml. A pharmaceutical composition may comprise an isotonic
agent. The isotonic agent may e.g. be selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid
(e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid,
tryptophan, threonine), an alditol (e.g. glycerol (glycerine),
1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)
polyethyleneglycol (e.g. PEG400), and mixtures thereof. Any sugar such as
mono-, di-, or polysaccharides, or water-soluble glucans, including for
example fructose, glucose, mannose, sorbose, xylose, maltose, lactose,
sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alfa and
beta HPCD, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. Sugar alcohol is defined as a
C4-C8 hydrocarbon having at least one --OH group and includes, for
example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and
arabitol. In one embodiment, the sugar alcohol additive is mannitol. A
pharmaceutical composition may comprise a chelating agent. The chelating
agent may e.g. be selected from salts of ethylenediaminetetraacetic acid
(EDTA), citric acid, and aspartic acid, and mixtures thereof. A
pharmaceutical composition may comprise a stabiliser. The stabiliser may
e.g. be one or more oxidation inhibitors, aggregation inhibitors,
surfactants, and/or one or more protease inhibitors. Non-limiting
examples of these various kinds of stabilisers are disclosed in the
following.

[0173] The term "aggregate formation" refers to a physical interaction
between the peptide molecules resulting in formation of oligomers, which
may remain soluble, or large visible aggregates that precipitate from the
solution. Aggregate formation by a peptide during storage of a liquid
pharmaceutical composition can adversely affect biological activity of
that polypeptide, resulting in loss of therapeutic efficacy of the
pharmaceutical composition. Furthermore, aggregate formation may cause
other problems such as blockage of tubing, membranes, or pumps when the
polypeptide-containing pharmaceutical composition is administered using
an infusion system.

[0174] A pharmaceutical composition may comprise an amount of an amino
acid base sufficient to decrease aggregate formation of the polypeptide
during storage of the composition. The term "amino acid base" refers to
one or more amino acids (such as methionine, histidine, imidazole,
arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), or
analogues thereof. Any amino acid may be present either in its free base
form or in its salt form. Any stereoisomer (i.e., L, D, or a mixture
thereof) of the amino acid base may be present.

[0175] Methionine (or other sulphuric amino acids or amino acid analogous)
may be added to inhibit oxidation of methionine residues to methionine
sulfoxide when the polypeptide acting as the therapeutic agent is a
polypeptide comprising at least one methionine residue susceptible to
such oxidation. Any stereoisomer of methionine (L or D) or combinations
thereof can be used.

[0176] A the pharmaceutical composition may comprise a stabiliser selected
from the group of high molecular weight polymers or low molecular
compounds. The stabiliser may e.g. be selected from polyethylene glycol
(e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,
carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L
and HPMC), cyclodextrins, sulphur-containing substances as
monothioglycerol, thioglycolic acid and 2-methylthioethanol, and
different salts (e.g. sodium chloride). A pharmaceutical composition may
comprise additional stabilising agents such as, but not limited to,
methionine and EDTA, which protect the polypeptide against methionine
oxidation, and a nonionic surfactant, which protects the polypeptide
against aggregation associated with freeze-thawing or mechanical
shearing.

[0177] A pharmaceutical composition may comprise one or more surfactants,
preferably a surfactant, at least one surfactant, or two different
surfactants. The term "surfactant" refers to any molecules or ions that
are comprised of a water-soluble (hydrophilic) part, and a fat-soluble
(lipophilic) part. The surfactant may e.g. be selected from the group
consisting of anionic surfactants, cationic surfactants, nonionic
surfactants, and/or zwitterionic surfactants.

[0178] A pharmaceutical composition may comprise one or more protease
inhibitors, such as, e.g., EDTA (ethylenediamine tetraacetic acid),
and/or benzamidineHCl.

[0180] Still further, a pharmaceutical composition may be formulated as is
known in the art of oral formulations of insulinotropic compounds, e.g.
using any one or more of the formulations described in WO 2008/145728.

[0181] An administered dose may contain from 0.01 mg-100 mg of the GLP-1
receptor agonist derivative, or from 0.01-50 mg, or from 0.01-20 mg, or
from 0.01-10 mg of the GLP-1 receptor agonist derivative.

[0182] The derivative may be administered in the form of a pharmaceutical
composition. It may be administered to a patient in need thereof at
several sites, for example, at topical sites such as skin or mucosal
sites; at sites which bypass absorption such as in an artery, in a vein,
or in the heart; and at sites which involve absorption, such as in the
skin, under the skin, in a muscle, or in the abdomen.

[0183] The route of administration may be, for example, lingual;
sublingual; buccal; in the mouth; oral; in the stomach; in the intestine;
nasal; pulmonary, such as through the bronchioles, the alveoli, or a
combination thereof; parenteral, epidermal; dermal; transdermal;
conjunctival; uretal; vaginal; rectal; and/or ocular. A composition may
be an oral composition, and the route of administration is per oral.

[0184] A composition may be administered in several dosage forms, for
example as a solution; a suspension; an emulsion; a microemulsion;
multiple emulsions; a foam; a salve; a paste; a plaster; an ointment; a
tablet; a coated tablet; a chewing gum; a rinse; a capsule such as hard
or soft gelatine capsules; a suppositorium; a rectal capsule; drops; a
gel; a spray; a powder; an aerosol; an inhalant; eye drops; an ophthalmic
ointment; an ophthalmic rinse; a vaginal pessary; a vaginal ring; a
vaginal ointment; an injection solution; an in situ transforming solution
such as in situ gelling, setting, precipitating, and in situ
crystallisation; an infusion solution; or as an implant. A composition
may be a tablet, optionally coated, a capsule, or a chewing gum.

[0185] A composition may further be compounded in a drug carrier or drug
delivery system, e.g. in order to improve stability, bioavailability,
and/or solubility. In a particular embodiment a composition may be
attached to such system through covalent, hydrophobic, and/or
electrostatic interactions. The purpose of such compounding may be, e.g.,
to decrease adverse effects, achieve chronotherapy, and/or increase
patient compliance.

[0186] A composition may also be used in the formulation of controlled,
sustained, protracting, retarded, and/or slow release drug delivery
systems.

[0187] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal, or intravenous injection by means of a
syringe, optionally a pen-like syringe, or by means of an infusion pump.

[0188] A composition may be administered nasally in the form of a
solution, a suspension, or a powder; or it may be administered pulmonally
in the form of a liquid or powder spray.

[0189] Transdermal administration is a still further option, e.g. by
needle-free injection, from a patch such as an iontophoretic patch, or
via a transmucosal route, e.g. buccally.

[0190] A composition may be a stabilised formulation. The term "stabilised
formulation" refers to a formulation with increased physical and/or
chemical stability, preferably both. In general, a formulation must be
stable during use and storage (in compliance with recommended use and
storage conditions) until the expiration date is reached.

[0191] The term "physical stability" refers to the tendency of the
polypeptide to form biologically inactive and/or insoluble aggregates as
a result of exposure to thermo-mechanical stress, and/or interaction with
destabilising interfaces and surfaces (such as hydrophobic surfaces). The
physical stability of an aqueous polypeptide formulation may be evaluated
by means of visual inspection, and/or by turbidity measurements after
exposure to mechanical/physical stress (e.g. agitation) at different
temperatures for various time periods. Alternatively, the physical
stability may be evaluated using a spectroscopic agent or probe of the
conformational status of the polypeptide such as e.g. Thioflavin T or
"hydrophobic patch" probes.

[0192] The term "chemical stability" refers to chemical (in particular
covalent) changes in the polypeptide structure leading to formation of
chemical degradation products potentially having a reduced biological
potency, and/or increased immunogenic effect as compared to the intact
polypeptide. The chemical stability can be evaluated by measuring the
amount of chemical degradation products at various time-points after
exposure to different environmental conditions, e.g. by SEC-HPLC, and/or
RP-HPLC.

[0194] The treatment with a derivative according to this invention may
also be combined with a surgery that influences the glucose levels,
and/or lipid homeostasis such as gastric banding or gastric bypass.

Pharmaceutical Indications

[0195] The present invention also relates to a GLP-1 receptor agonist
peptide of the invention, and a derivative thereof, for use as a
medicament.

[0196] In particular embodiments, these compounds may be used for the
following medical treatments, all preferably relating one way or the
other to diabetes:

[0205] (x) prevention and/or treatment of critical illness, such as
treatment of a critically ill patient, a critical illness
poly-nephropathy (CIPNP) patient, and/or a potential CIPNP patient;
prevention of critical illness or development of CIPNP; prevention,
treatment and/or cure of systemic inflammatory response syndrome (SIRS)
in a patient; and/or for the prevention or reduction of the likelihood of
a patient suffering from bacteraemia, septicaemia, and/or septic shock
during hospitalisation; and/or

[0207] In a particular embodiment, the indication is selected from the
group consisting of (i)-(iii) and (v)-(iix), such as indications (i),
(ii), and/or (iii); or indication (v), indication (vi), indication (vii),
and/or indication (iix).

[0208] In another particular embodiment, the indication is (i). In a
further particular embodiment the indication is (v). In a still further
particular embodiment the indication is (iix).

[0226] 11. The peptide of any one of embodiments 1-10, wherein Y is Chem.
4. 12. The peptide of any one of embodiments 1-11, wherein X1 is N.
13. The peptide of any one of embodiments 1-12, wherein one of X2,
X3, X4, and X5 is N. 14. The peptide of any one of
embodiments 1-13, preferably the peptide of embodiment 12, wherein i)
X3 is N; ii) X4 is N; iii) X5 is N; or X2 is N. 15.
The peptide of embodiment any one of embodiments 1-14, wherein R11 and
R12 independently represent hydrogen, alkyl, aryl, halogen, hydroxyl,
hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy,
aryloxy, carboxamide, alkyl ester, or aryl ester; wherein preferably
alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy, and/or alkyl
ester contains lower alkyl, straight or branched, more preferably having
1-6 C-atoms. 16. The peptide of any one of embodiment 1-15, wherein R11
and R12 independently represent hydrogen, lower alkyl, or lower alkoxy,
wherein the lower alkyl and lower alkoxy, independently, have 1-5
C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms. 17. The
peptide of any one of embodiments 1-16, wherein R11 and R12 independently
represent hydrogen, alkyl having 1-2 C-atoms (ethyl, or methyl), or
alkoxy having 1-2 C-atoms (ethoxy, methoxy). 18. The peptide of any one
of embodiments 1-17, wherein R11 and R12 are methyl or hydrogen,
preferably hydrogen. 19. The peptide of any one of embodiments 1-18,
wherein Y is a derivative of 1H-imidazole, preferably 1H-imidazol-4-yl,
optionally substituted at one or two of positions 2, 3, and/or 5, wherein
the position numbering of imidazole is according to IUPAC, and/or as
shown for imidazole on wikipedia on 3 Dec. 2010 at 18:00 DK time. 20. The
peptide of any one of embodiments 1-19, wherein Q is attached to i)
X2, ii) X3, X4, or X5, preferably to X2 or
X4, most preferably to X4. 21. The peptide of any one of
embodiments 1-20, wherein q is 1-5, preferably 1-4, more preferably 1-3,
even more preferably 1, 2, or 3. 22. The peptide of any one of
embodiments 1-21, wherein R15 and R16 independently of each other and
independently for each value of q represent hydrogen. 23. The peptide of
any one of embodiments 1-22, wherein R15 and R16 both represent hydrogen.
24. The peptide of any one of embodiments 1-23, wherein R is hydrogen.
25. The peptide of any one of embodiments 1-24, wherein Y is a derivative
of an imidazole, such as 1H-imidazol, being substituted with a group of
formula Chem. 7:

*--(R17)--N--*, Chem. 7

wherein R17 represents alkylene, straight or branched, having 1-6
C-atoms, preferably 1-5 C-atoms, more preferably 1-4 C-atoms, or most
preferably 1-3 C-atoms. 26. The peptide of any one of embodiments 1-10,
wherein Y is Chem. 5.

28. The peptide of any one of embodiment 1-10 and 26-27, wherein R13 and
R14 independently represent hydrogen, lower alkyl, or lower alkoxy,
wherein the lower alkyl and lower alkoxy, independently, have 1-5
C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms. 29. The
peptide of any one of embodiments 1-10 and 26-28, wherein R13 and R14
independently represent hydrogen, alkyl having 1-2 C-atoms (ethyl, or
methyl), or alkoxy having 1-2 C-atoms (ethoxy, methoxy). 30. The peptide
of any one of embodiments 1-10 and 26-29, wherein R13 and R14 are methyl
or hydrogen, preferably hydrogen. 31. The peptide of any one of
embodiments 1-10 and 26-30, wherein Y is a derivative of pyridine,
preferably pyridin-2-yl, optionally substituted at one or two of
positions 3, 4, 5, and/or 6, where the position numbering of pyridine is
according to IUPAC, and/or as shown for pyridine on wikipedia on 3 Dec.
2010 at 18:00 DK time. 32. The peptide of any one of embodiments 1-10 and
26-31, wherein Q is attached to position 2, 3, 4, 5, or 6 of the pyridine
ring, preferably to position 2, where the position numbering of pyridine
is as defined in embodiment 31. 33. The peptide of any one of embodiments
1-32, wherein q is as defined in embodiment 21. 34. The peptide of any
one of embodiments 1-33, wherein R15 and R16 are as defined in any one of
embodiments 22-23. 35. The peptide of any one of embodiments 1-34,
wherein R is hydrogen. 36. The peptide of any one of embodiments 1-35,
wherein the GLP-1 receptor agonist peptide of which P is a fragment
lacking the two N-terminal amino acid residues is an agonist of the human
GLP-1 receptor, wherein agonist activity is preferably determined as
stimulation of the formation of cAMP in a medium containing the human
GLP-1 receptor. 37. The peptide of embodiment 36, wherein the medium is a
suitable medium, such as a medium containing the human GLP-1 receptor and
having the following composition (final in-assay concentrations): 50 mM
Tris-HCl, 1 mM EGTA, 1.5 mM MgSO4, 1.7 mM ATP, 20 mM GTP, 2 mM
3-isobutyl-1-methylxanthine (IBMX), 0.01% Tween-20, pH 7.4; more
preferably the following composition (final in-assay concentrations): 50
mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl2, 6H2O; 150 mM NaCl; 0.01%
Tween; 0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP; pH 7.4. 38. The peptide
of any one of embodiments 1-37, wherein the GLP-1 receptor agonist
peptide of which P is a fragment lacking the two N-terminal amino acid
residues has a potency corresponding to an EC50 of below 1000 pM,
preferably below 500 pM, more preferably below 250 pM, even more
preferably below 125 pM, or most preferably below 50 pM. 39. The peptide
of any one of embodiments 1-38, wherein the GLP-1 receptor agonist
peptide of which P is a fragment lacking the two N-terminal amino acid
residues is selected from His-Ala-"P", His-Gly-"P", and "His-Ser-P". 40.
The peptide of any one of embodiments 1-39, wherein the GLP-1 receptor
agonist peptide of which P is a fragment lacking the two N-terminal amino
acid residues is selected from His-Ala-"P" and His-Gly-"P". 41. The
peptide of any one of embodiments 1-40, wherein P is selected from i)
GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), iii)
GLP-1A(3-37) (SEQ ID NO: 3), and iv) analogues of i), ii), or iii) having
a maximum of eight amino acid residues exchanged as compared to the
respective sequence i), ii), or iii) with which the analogue has the
highest similarity, or, preferably, percentage of identity. 42. The
peptide of any one of embodiments 1-41, wherein P is GLP-1(9-37) (SEQ ID
NO: 1), or an analogue thereof having a maximum of eight amino acid
residues exchanged as compared to SEQ ID NO: 1. 43. The peptide of any
one of embodiments 1-42, wherein P has a) a maximum of seven, six, or
five; preferably b) a maximum of four, or c) most preferably a maximum of
three amino acid residues exchanged, as compared to one of the sequences
of i) GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and
iii) GLP-1A(3-37) (SEQ ID NO: 3), with which P has the highest
similarity, wherein preferably the comparison is made with GLP-1(9-37)
(SEQ ID NO: 1), or with GLP-1A(3-37), most preferably with GLP-1(9-37).
44. The peptide of any one of embodiments 1-43, wherein P has a maximum
of two, preferably a maximum of one, or most preferably no amino acid
residues exchanged as compared to one of the sequences of i) GLP-1(9-37)
(SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii) GLP-1A(3-37)
(SEQ ID NO: 3), with which P has the highest similarity, wherein
preferably the comparison is made with GLP-1(9-37) (SEQ ID NO: 1), or
with GLP-1A(3-37), most preferably with GLP-1(9-37). 45. The peptide of
any one of embodiments 1-44, which has a C-terminal amide. 46. The
peptide of any one of embodiments 1-45, which has a C-terminal --COOH
group. 47. The peptide of any one of embodiments 1-46, which comprises at
least one of the following substitutions as compared to GLP-1(9-37) (SEQ
ID NO: 1): 18K; 22E; 30E; 31H; 34Q,R; 36K; 37K; and/or 38E. 48. The
peptide of any one of embodiments 1-47, which comprises 18K. 49. The
peptide of any one of embodiments 1-48, which comprises 22E. 50. The
peptide of any one of embodiments 1-49, which comprises 30E. 51. The
peptide of any one of embodiments 1-50, which comprises 31H. 52. The
peptide of any one of embodiments 1-51, which comprises 34Q or 34R,
preferably 34R. 53. The peptide of any one of embodiments 1-52, which
comprises 36K. 54. The peptide of any one of embodiments 1-53, which
comprises 37K. 55. The peptide of any one of embodiments 1-54, which
comprises 38E. 56. The peptide of any one of embodiments 1-55, which
comprises 34R and 37K. 57. The peptide of any one of embodiments 1-56,
which comprises 30E and 36K. 58. The peptide of any one of embodiments
1-57, which comprises 31H and 34Q. 59. The peptide of any one of
embodiments 56-57, which further comprises 38E. 60. The peptide of any
one of embodiments 1-59 which comprises the following substitutions: (i)
18K, 22E, 34Q; (ii) 31H, 34Q; (iii) 30E, 36K; (iv) 30E, 36K, 38E; (v)
34R; (vi) 34R, 37K; or (vii) 34R, 37K, 38E. 61. The peptide of any one of
embodiments 1-60, which has the following substitutions as compared to
GLP-1(9-37) (SEQ ID NO: 1), all other amino acid residues being as in SEQ
ID NO: 1: (i) 18K, 22E, 34Q; (ii) 31H, 34Q; (iii) 30E, 36K; (iv) 30E,
36K, 38E; (v) 34R; (vi) 34R, 37K; of (vii) 34R, 37K, 38E. 62. The peptide
of any one of embodiments 1-46, which comprises at least one of the
following substitutions as compared to GLP-1A(3-37) (SEQ ID NO: 3):
17Q,R; 20R; 33R; and/or 38K. 63. The peptide of any one of embodiments
1-46, and 62, which has the following substitutions, as compared to
GLP-1A(3-37) (SEQ ID NO: 3), all other amino acid residues being as in
SEQ ID NO: 3: 17R, 20R, 33R, and 38K. 64. A GLP-1 receptor agonist
peptide selected from the following: [0228] (i)
N9-[-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-Ly-
s18,Glu22,Gln34]GLP-1(9-37)-peptide; [0229] (ii)
N9-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[His-
31,Gln34]GLP-1 (9-37)-peptide; [0230] (iii)
N9-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Glu-
30,Lys36]GLP-1(9-37)-Glu38-peptide amide; [0231] (iv)
N9-{2-[2-(1H-Imidazol-4-yl)-propylcarbamoyl]-2-methyl-propionyl}-Glu-
30,Lys36]GLP-1 (9-37)Glu38-peptide amide; [0232] (v)
N9-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Gl-
u30,Lys36]GLP-1(9-37)-Glu38-peptide amide; [0233] (vi)
N9-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Ar-
g17,Arg20,Arg33,Lys38]GLP-1A(3-37)-peptide; [0234]
(vii) N9-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl-
}[Arg34,Lys37]GLP-1(9-37)-peptide; [0235] (iix)
N9-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Arg-
34, Lys37]GLP-1 (9-37)Glu38-peptide; [0236] (ix)
N9-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Arg-
34, Lys37]GLP-1 (9-37)-peptide; [0237] (x)
N9-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}[Arg.-
sup.3]GLP-1(9-37)-peptide; and [0238] (xii)
N9-[2,2-dimethyl-3-oxo-3-(pyridin-2-ylmethylamino)propanoyl][Arg.sup-
.34,Lys37]-GLP-1-(9-37)-peptide; or a pharmaceutically acceptable
salt, amide, or ester thereof. 64. The peptide of any one of embodiments
1-63 which has a maximum of two K residues. 65. The peptide of any one of
embodiments 1-64, which has a maximum of one K residue. 66. The peptide
of any one of embodiments 1-65, wherein

68. The peptide of embodiment 67, wherein the alignment program is a
Needleman-Wunsch alignment. 69. The peptide of any one of embodiments
67-68, wherein the default scoring matrix and the default identity matrix
is used. 70. The peptide of any one of embodiments 67-69, wherein the
scoring matrix is BLOSUM62. 71. The peptide of any one of embodiments
67-70, wherein the penalty for the first residue in a gap is -10 (minus
ten). 72. The peptide of any one of embodiments 67-71, wherein the
penalties for additional residues in a gap is -0.5 (minus point five).
73. The peptide of any one of embodiments 1-72, wherein, a residue
number, preferably any residue number, be it in superscript after an
amino acid residue, or in ordinary script before or after the amino acid
residue in question, refers to the corresponding position in one of the
sequences of i) GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID
NO: 2), and iii) GLP-1A(3-37) (SEQ ID NO: 3). 74. A derivative of a
peptide of any one of embodiments 1-73, or a pharmaceutically acceptable
salt, amide, or ester thereof. 75. The derivative of embodiment 74 which
comprises, preferably has, an albumin binding moiety attached to a lysine
residue, more preferably to the epsilon-amino group thereof, via an amide
bond. 76. The derivative of embodiment 75, which comprises, preferably
has, an albumin binding moiety attached to one or more of 18K, 26K, 36K,
and/or 37K; preferably one albumin binding moiety attached to 18K, 26K,
or 36K; or two albumin binding moieties attached to 26K and 37K; wherein
reference may be had to the sequence of GLP-1(9-37) (SEQ ID NO: 1). 77.
The derivative of embodiment 75, which has an albumin binding moiety
attached to one or more of 17K, 20K, 33K, and/or 38K; preferably one
albumin binding moiety attached to 38K; or two albumin binding moieties
attached to 20K and 33K; wherein reference may be had to the sequence of
GLP-1A(3-37) (SEQ ID NO: 3). 78. The derivative of any one of embodiments
75-77, in which the albumin binding moiety comprises a protracting
moiety. 79. The derivative of embodiment 78, wherein

[0244] in which x is an integer in the range of 6-18, y is an integer in
the range of 3-17, z is an integer in the range of 1-5, and R18 is a
group having a molar mass not higher than 150 Da.

80. The derivative of embodiment 79, in which the protracting moiety is
Chem. 8, in which x is an even number, preferably in the range of 14-18,
more preferably 16-18, or most preferably x is 16. 81. The derivative of
embodiment 79, in which the protracting moiety is Chem. 9, in which y is
an odd number, preferably in the range of 7-15. 82. The derivative of
embodiment 79, in which the protracting moiety is Chem. 10, in which z is
an odd number, preferably 3; and R18 is tert. butyl. 83. The
derivative of any one of embodiments 79 and 81, in which y is 9. 84. The
derivative of any one of embodiments 79, 81, and 83, in which the --COOH
group is in the para-position. 85. The derivative of any one of
embodiments 79, and 82, in which R18 is in the para-position. 86.
The derivative of any one of embodiments 75-84 which comprises a linker.
87. The derivative of any one of embodiments 75-86, wherein the albumin
binding moiety comprises a linker. 88. The derivative of any one of
embodiments 75-87, wherein the albumin binding moiety further comprises a
linker. 89. The derivative of any one of embodiments 86-88, wherein the
linker is a di-radical which comprises an N radical and a CO radical,
wherein i) the N-radical is represented by a first *--NR19R20
group, where R19 and R20 may, independently, designate
hydrogen, carbon, or sulphur, optionally substituted; and ii) the CO
radical is represented by a first *--CO group, and wherein, preferably,
the first *--NR19R20 group is capable of forming an amide bond
with a second *--CO group, and the first *--CO group is capable of
forming an amide bond with a second *--NR19R20 group, wherein
the second *--NR19R20 group and the second *--CO group are
defined as the first *--NR19R20 group and the first *--CO
group, respectively, and form part, independently, of the structure of i)
the analogue, ii) the protracting moiety, and/or iii) another linker. 90.
The derivative of any one of embodiments 74-89, which comprises at least
one linker selected from the group consisting of Chem. 11, Chem. 12,
Chem. 13, and Chem. 14:

*--NH--CH2--CH2--(O--CH2--CH2)k--O--(CH2).-
sub.n--CO--* Chem. 11

*--NH--C(COOH)--(CH2)2--CO--* Chem. 12

*--N--C((CH2)2COOH)--CO--* Chem. 13

*--NC5H8--CO--* Chem. 14

wherein k is an integer in the range of 1-5, and n is an integer in the
range of 1-5; and wherein Chem. 12 and Chem. 13 are di-radicals of Glu.
91. The derivative of embodiment 90, wherein the linker comprises Chem.
11, and wherein preferably Chem. 11 is a first linker element. 92. The
derivative of any one of embodiments 90-91, wherein k is 1. 93. The
derivative of any one of embodiments 90-92, wherein n is 1. 94. The
derivative of any one of embodiments 90-93, wherein Chem. 11 is included
m times, wherein m is an integer in the range of 1-10. 95. The derivative
of embodiment 94, wherein m is an integer in the range of 1-6; preferably
in the range of 1-4; more preferably m is 1 or 2; even more preferably m
is 1; or most preferably m is 2. 96. The derivative of any one of
embodiments 94-95, wherein, when m is different from 1, the Chem. 11
elements are interconnected via amide bond(s). 97. The derivative of any
one of embodiments 90-96, wherein the linker consists of one or more
Chem. 11 elements. 98. The derivative of any one of embodiments 90-97,
wherein Chem. 11 is represented by Chem. 11a:

##STR00019##

wherein k and n are as defined in any one of embodiments 90-97. 99. The
derivative of any one of embodiments 90-99, wherein the linker comprises
a Glu di-radical, such as Chem. 12, and Chem. 13. 100. The derivative of
any one of embodiments 90-99, wherein Chem. 12 and Chem. 13,
independently, may be represented by Chem. 12a and Chem. 13a,
respectively:

##STR00020##

most preferably by Chem. 12a. 101. The derivative of any one of
embodiments 90-101, wherein the Glu di-radical, such as Chem. 12, and/or
Chem. 13, independently, is included p times, wherein p is an integer in
the range of 1-3. 102. The derivative of embodiment 101, wherein p is 1,
2, or 3; preferably 1 or 2, or most preferably 1. 103. The derivative of
any one of embodiments 90-102, wherein the Glu di-radical is a radical of
L-Glu or D-Glu, preferably of L-Glu. 104. The derivative of any one of
embodiments 90-103, wherein the linker consists of a Glu di-radical,
preferably Chem. 12, more preferably Chem. 12a. 105. The derivative of
any one of embodiments 90-104, wherein the linker comprises Chem. 14.
106. The derivative of any one of embodiments 90-105, where Chem. 14 is
represented by Chem. 14a:

##STR00021##

107. The derivative of any one of embodiments 90-106, wherein the linker
consists of Chem. 11, being connected at its *--NH end to * the *--CO end
of the protracting moiety, and at its *--CO end to the epsilon amino
group of a lysine residue of the peptide. 108. The derivative of any one
of embodiments 90-106, wherein the linker consists of one time Chem. 12
and two times Chem. 11, interconnected via amide bonds and in the
sequence indicated, the linker being connected at its *--NH end to the
*--CO end of the protracting moiety, and at its *--CO end to the epsilon
amino group of a lysine residue of the peptide. 109. The derivative of
any one of embodiments 90-106, wherein the linker consists of one time
Chem. 14, one time Chem. 12, and two times Chem. 11, interconnected via
amide bonds and in the sequence indicated, the linker being connected at
its *--NH end to the *--CO end of the protracting moiety, and at its
*--CO end to the epsilon amino group of a lysine residue of the peptide.
110. The derivative of any one of embodiments 90-109, wherein the one or
more linker(s) are interconnected via amide bond(s). 111. A compound
selected from the following: Chem. 30, Chem. 31, Chem. 32, Chem. 33,
Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem. 39, and Chem. 41;
or a pharmaceutically acceptable salt, amide, or ester thereof. 112. A
compound characterised by its name, and selected from a listing of each
of the names of the compounds of Examples 1-10, and 12 herein; or a
pharmaceutically acceptable salt, amide, or ester thereof. 113. The
compound of embodiment 112, which is a compound of embodiment 111. 114.
The compound of any one of embodiments 111-113, which is a derivative of
any one of embodiments 74-110. 115. The peptide or derivative of any one
of embodiments 1-114, which has GLP-1 activity. 116. The peptide or
derivative of embodiment 115, wherein GLP-1 activity refers to the
capability of activating the human GLP-1 receptor. 117. The peptide or
derivative of embodiment 116, wherein activation of the human GLP-1
receptor is measured in an in vitro assay, as the potency of cAMP
production. 118. The peptide or derivative of any one of embodiments
1-117, which has a potency corresponding to an EC50 at or below 4500
pM, preferably below 4500 pM, more preferably below 4000 pM, even more
preferably below 3500 pM, or most preferably below 3000 pM. 119. The
peptide or derivative of any one of embodiments 1-118, which has a
potency corresponding to an EC50 below 2500 pM, preferably below
2000 pM, more preferably below 1500 pM, even more preferably below 1000
pM, or most preferably below 800 pM. 120. The peptide or derivative of
any one of embodiments 1-119 which has a potency corresponding to an
EC50 below 600 pM, preferably below 500 pM, more preferably below
400 pM, even more preferably below 300 pM, or most preferably below 200
pM. 121. The peptide or derivative of any one of embodiments 1-120 which
has a potency corresponding to an EC50 below 180 pM, preferably
below 160 pM, more preferably below 140 pM, even more preferably below
120 pM, or most preferably below 100 pM. 122. The peptide or derivative
of any one of embodiments 1-121 which has a potency corresponding to an
EC50 below 80 pM, preferably below 60 pM, more preferably below 50
pM, even more preferably below 40 pM, or most preferably below 30 pM.
123. The peptide or derivative of any one of embodiments 1-122, wherein
the potency is determined as EC50 for the dose-response curve
showing dose-dependent formation of cAMP in a medium containing the human
GLP-1 receptor, preferably using a stable transfected cell-line such as
BHK467-12A (tk-ts13), and/or using for the determination of cAMP a
functional receptor assay, e.g. based on competition between endogenously
formed cAMP and exogenously added biotin-labelled cAMP, in which assay
cAMP is more preferably captured using a specific antibody, and/or
wherein an even more preferred assay is the AlphaScreen cAMP Assay, most
preferably the one described in Example 13. 124. The peptide or
derivative of any one of embodiments 1-123, the EC50 of which is
less than 10 times the EC50 of semaglutide, preferably less than 8
times the EC50 of semaglutide, more preferably less than 6 times the
EC50 of semaglutide, even more preferably less than 4 times the
EC50 of semaglutide, or most preferably less than 2 times the
EC50 of semaglutide. 125. The peptide or derivative of any one of
embodiments 1-124, the EC50 of which is less than the EC50 of
semaglutide, preferably less than 0.8 times the EC50 of semaglutide,
more preferably less than 0.6 times the potency of semaglutide, even more
preferably less than 0.4 times the EC50 of semaglutide, or most
preferably less than 0.2 times the EC50 of semaglutide. 126. The
peptide or derivative of any one of embodiments 1-125, the EC50 of
which is less than 10 times the EC50 of liraglutide, preferably less
than 8 times the EC50 of liraglutide, more preferably less than 6
times the EC50 of liraglutide, even more preferably less than 4
times the EC50 of liraglutide, or most preferably less than 2 times
the EC50 of liraglutide. 127. The peptide or derivative of any one
of embodiments 1-126, the EC50 of which is less than the EC50
of liraglutide, preferably less than 0.8 times the EC50 of
liraglutide, more preferably less than 0.6 times the EC50 of
liraglutide, even more preferably less than 0.5 times the EC50 of
liraglutide, or most preferably less than or at 0.4 times the EC50
of liraglutide. 128. The derivative of any one of embodiments 74-127, for
which the ratio [GLP-1 receptor binding affinity (IC50) in the
presence of 2.0% HSA (high albumin), divided by GLP-1 receptor binding
affinity (IC50) in the presence of 0.005% HSA (low albumin)] is: a)
at least 0.5, preferably at least 1.0, more preferably at least 10, even
more preferably at least 20, or most preferably at least 30; b) at least
40, preferably at least 50, more preferably at least 60, even more
preferably at least 70, or most preferably at least 80; c) at least 90,
preferably at least 100, more preferably at least 110, or most preferably
at least 120; d) at least 20% of the ratio of semaglutide, preferably at
least 50% of the ratio of semaglutide, more preferably at least 75% of
the ratio of semaglutide, even more preferably at least equal to the
ratio of semaglutide, or most preferably at least twice the ratio of
semaglutide; or e) at least equal to the ratio of liraglutide, preferably
at least twice the ratio of liraglutide, more preferably at least three
times the ratio of liraglutide, even more preferably at least 5 times the
ratio of liraglutide, or most preferably at least 10 times the ratio of
liraglutide. 129. The derivative of any one of embodiments 74-128, for
which the GLP-1 receptor binding affinity (IC50) in the presence of
0.005% HSA (low albumin) is a) below 600.00 nM, preferably below 500.00
nM, more preferably below 200.00 nM, even more preferably below 100.00
nM, or most preferably below 45.00 nM; or b) below 20.00 nM, preferably
below 10.00 nM, more preferably below 5.00 nM, even more preferably below
2.00 nM, or most preferably below 1.00 nM. 130. The derivative of any one
of embodiments 1-129, for which the GLP-1 receptor binding affinity
(IC50) in the presence of 2.0% HSA (high albumin) is a) below 900
nM, more preferably below 800 nM, even more preferably below 700 nM, or
most preferably below 600 nM; or b) below 400.00 nM, preferably below
300.00 nM, more preferably below 200.00 nM, even more preferably below
100.00 nM, or most preferably below 50.00 nM. 131. The derivative of any
one of embodiments 1-130, wherein the binding affinity to the GLP-1
receptor is measured by way of displacement of 125I-GLP-1 from the
receptor, preferably using a SPA binding assay. 132. The derivative of
any one of embodiments 1-131, wherein the GLP-1 receptor is prepared
using a stable, transfected cell line, preferably a hamster cell line,
more preferably a baby hamster kidney cell line, such as BHK tk-ts13.
133. The analogue or derivative of any one of embodiments 1-132, wherein
the IC50 value is determined as the concentration which displaces
50% of 125I-GLP-1 from the receptor. 134. The derivative of any one
of embodiments 74-133, which has an oral bioavailability, preferably an
absolute oral bioavailability, which is higher than that of liraglutide;
and/or higher than that of semaglutide. 135. The derivative of embodiment
134, wherein oral bioavailability is measured in vivo in rats, as
exposure in plasma after direct injection into the intestinal lumen. 136.
The derivative of any one of embodiments 74-135, for which the plasma
concentration (pM) of the derivative, determined 30 minutes after
injection of a solution of the derivative in the jejunum of rat, divided
by the concentration (pM) of the injected solution (dose-corrected
exposure at 30 min) is at least 15, preferably at least 30, more
preferably at least 48, still more preferably at least 62, even more
preferably at least 80, or most preferably at least 100. 137. The
derivative of any one of embodiments 74-136, for which the plasma
concentration (pM) of the derivative, determined 30 minutes after
injection of a solution of the derivative in the jejunum of rat, divided
by the concentration (pM) of the injected solution (dose-corrected
exposure at 30 min) is at least 30, preferably at least 40, more
preferably at least 50, still more preferably at least 60, even more
preferably at least 70, or most preferably at least 80. 138. The
derivative of any one of embodiments 74-137, wherein the Derivative is
tested in a concentration of 1000 uM in admixture with 55 mg/ml sodium
caprate. 139. The derivative of any one of embodiments 74-138, wherein
male Sprague Dawley rats are used, preferably with a body weight upon
arrival of approximately 240 g. 140. The derivative of any one of
embodiments 74-139, wherein the rats are fasted for approximately 18
hours before the experiment. 141. The derivative of any one of
embodiments 74-140, wherein the rats are taken into general anaesthesia
after having fasted and before the injection of the derivative in the
jejunum. 142. The derivative of any one of embodiments 74-141, wherein
the derivative is administered in the proximal part of the jejunum (10 cm
distal for the duodenum) or in the mid-intestine (50 cm proximal for the
cecum). 143. The derivative of any one of embodiments 74-142, wherein 100
μl of the derivative is injected into the jejunal lumen through a
catheter with a 1 ml syringe, and subsequently 200 μl of air is pushed
into the jejunal lumen with another syringe, which is then left connected
to the catheter to prevent flow back into the catheter. 144. The
derivative of any one of embodiments 74-143, wherein blood samples (200
ul) are collected into EDTA tubes from the tail vein at desired
intervals, such as at times 0, 10, 30, 60, 120 and 240 min, and
centrifuged 5 minutes, 10000G, at 4° C. within 20 minutes. 145.
The derivative of any one of embodiments 74-144, wherein plasma (75 ul)
is separated, immediately frozen, and kept at -20° C. until
analyzed for plasma concentration of the derivative. 146. The derivative
of any one of embodiments 74-145, wherein LOCI (Luminescent Oxygen
Channeling Immunoassay) is used for analyzing the plasma concentration of
the derivative. 147. The derivative of any one of embodiments 74-145,
which has a more protracted profile of action than liraglutide. 148. The
derivative of embodiment 147, wherein protraction means half-life in vivo
in a relevant animal species, such as db/db mice, rat, pig, and/or,
preferably, minipig; wherein the derivative is administered i) s.c.,
and/or, preferably, ii) s.c. 149. The derivative of any one of
embodiments 74-148, wherein the terminal half-life (T1/2) after i.v.
administration in minipigs is a) at least 12 hours, preferably at least
24 hours, more preferably at least 36 hours, even more preferably at
least 48 hours, or most preferably at least 60 hours; or b) at least 0.2
times the half-life of semaglutide, preferably at least 0.4 times the
half-life of semaglutide, more preferably at least 0.6 times the
half-life of semaglutide, even more preferably at least 0.8 times the
half-life of semaglutide, or most preferably at least the same as the
half-life of semaglutide. 150. The derivative of embodiment 149, wherein
the minipigs are male Gottingen minipigs. 151. The derivative of any one
of embodiments 149-150, wherein the minipigs are 7-14 months of age, and
preferably weighing from 16-35 kg. 152. The derivative of any one of
embodiments 149-151, wherein the minipigs are housed individually, and
fed once or twice daily, preferably with SDS minipig diet. 153. The
derivative of any one of embodiments 149-152, wherein the derivative is
dosed, i.v., after at least 2 weeks of acclimatisation. 154. The
derivative of any one of embodiments 149-153, wherein the animals are
fasted for approximately 18 h before dosing and for at least 4 h after
dosing, and have ad libitum access to water during the whole period. 155.
The derivative of any one of embodiments 149-154, wherein the Derivative
is dissolved in 50 mM sodium phosphate, 145 mM sodium chloride, 0.05%
tween 80, pH 7.4 to a suitable concentration, preferably from 20-60
nmol/ml. 156. The derivative of any one of embodiments 149-155, wherein
intravenous injections of the derivative are given in a volume
corresponding to 1-2 nmol/kg. 157. The derivative of any one of
embodiments 74-156, which is not the compound of Example 2, preferably
not Chem. 31. 158. The derivative of any one of embodiments 74-157, which
is not the compound of Examples 7, 8, 9, and 12; preferably not Chem. 36,
Chem. 37, Chem. 38, and Chem. 41. 158. A peptide according to any one of
embodiments 1-73 and 115-127, for use as a medicament. 159. A derivative
according to any one of embodiments 74-157, for use as a medicament. 160.
A peptide according to any one of embodiments 1-73 and 115-127, for use
in the treatment and/or prevention of all forms of diabetes and related
diseases, such as eating disorders, cardiovascular diseases,
gastrointestinal diseases, diabetic complications, critical illness,
and/or polycystic ovary syndrome; and/or for improving lipid parameters,
improving β-cell function, and/or for delaying or preventing
diabetic disease progression. 161. A derivative according to any one of
embodiments 74-157, for use in the treatment and/or prevention of all
forms of diabetes and related diseases, such as eating disorders,
cardiovascular diseases, gastrointestinal diseases, diabetic
complications, critical illness, and/or polycystic ovary syndrome; and/or
for improving lipid parameters, improving β-cell function, and/or
for delaying or preventing diabetic disease progression. 162. Use of a
peptide according to any one of embodiments 1-73 and 115-127 in the
manufacture of a medicament for treatment and/or prevention of all forms
of diabetes and related diseases, such as eating disorders,
cardiovascular diseases, gastrointestinal diseases, diabetic
complications, critical illness, and/or polycystic ovary syndrome; and/or
for improving lipid parameters, improving β-cell function, and/or
for delaying or preventing diabetic disease progression. 163. Use of a
derivative according to any one of embodiments 74-157, in the manufacture
of a medicament for treatment and/or prevention of all forms of diabetes
and related diseases, such as eating disorders, cardiovascular diseases,
gastrointestinal diseases, diabetic complications, critical illness,
and/or polycystic ovary syndrome; and/or for improving lipid parameters,
improving β-cell function, and/or for delaying or preventing
diabetic disease progression. 164. A method of treating or preventing all
forms of diabetes and related diseases, such as eating disorders,
cardiovascular diseases, gastrointestinal diseases, diabetic
complications, critical illness, and/or polycystic ovary syndrome; and/or
for improving lipid parameters, improving β-cell function, and/or
for delaying or preventing diabetic disease progression, by administering
a pharmaceutically active amount of a peptide according to any one of
embodiments 1-73 and 115-127. 165. A method of treating or preventing all
forms of diabetes and related diseases, such as eating disorders,
cardiovascular diseases, gastrointestinal diseases, diabetic
complications, critical illness, and/or polycystic ovary syndrome; and/or
for improving lipid parameters, improving β-cell function, and/or
for delaying or preventing diabetic disease progression, by administering
a pharmaceutically active amount of a derivative according to any one of
embodiments 74-157. 166. An intermediate product of the formula Chem. 50
or Chem. 51:

##STR00022##

wherein Q represents a bond, or a group of formula Chem. 6:

--(C(R15)(R16))q--, Chem. 6

wherein q is 1-6, and R15 and R16 independently of each other and
independently for each value of q represent hydrogen, alkyl, carboxyl, or
hydroxyl; R represents hydrogen, or alkyl; R1 and R2 independently
represent (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen,
hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl,
carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide,
alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl, or (ii) R1 and
R2 together form cyclo alkyl, heterocyclyl, or heteroaryl; and each of
PG1 and PG2 represents a protection group; with the optional
proviso (iii) that R1 and R2 do not both represent hydrogen; or a
pharmaceutically acceptable salt, ester, or amide thereof. 167. The
intermediate product of embodiment 166, wherein PG1 is a group that
reversibly renders the N-atom to which it is attached unreactive, and
that can be removed selectively. 168. The intermediate product of any one
of embodiments 165-167, wherein PG1 is selected from the group
consisting of Boc, Trt, Mtt, Bzl, Tos, Clt, Mmt, Bom, and Fmoc. 169. The
intermediate product of any one of embodiments 166-168, wherein PG2
is a group that reversibly renders the --CO group to which it is attached
unreactive, and that can be removed selectively. 170. The intermediate
product of any one of embodiments 165-169, wherein PG2 is i) --OH,
or ii) functionalised as an activated ester. 171. The intermediate
product of embodiment 170, wherein the activated ester is an ester of
p-nitrophenol; 2,4,5-trichlorophenol; N-hydroxysuccinimide;
N-hydroxysulfosuccinimide;
3,4-dihydro-3-hydroxy-1,2,3-benzotriazine-4-one;
5-chloro-8-hydroxyquinoline; N-hydroxy-5-norbornene-2,3-dicarboxylic acid
imide; pentafluorophenol; p-sulfotetrafluorophenol; N-hydroxyphthalimide;
1-hydroxybenzotriazole; 1-hydroxy-7-azabenzotriazole; N-hydroxymaleimide;
4-hydroxy-3-nitrobenzene sulfonic acid; or any other activated ester
known in the art. 172. The intermediate product of any one of embodiments
165-171, wherein PG2 is selected from OPfp, OPnp, and OSuc. 173. The
intermediate product of any one of embodiments 166-172, wherein, PG1
is Trt. 174. The intermediate product of any one of embodiments 166-173,
wherein q is 1-5, preferably 1-4, more preferably 1-3. 175. The
intermediate product of any one of embodiments 166-174, wherein R15 and
R16 independently of each other and independently for each value of q
represent hydrogen. 176. The intermediate product of any one of
embodiments 166-175, wherein R15 and R16 both represent hydrogen. 177.
The intermediate product of any one of embodiments 166-176, wherein Q is
--(CH2)n, wherein n is 1, 2, or 3. 178. The intermediate
product of any one of embodiments 166-177, wherein R is hydrogen. 179.
The intermediate product of any one of embodiments 166-178, wherein R1
and R2 independently represent hydrogen, alkyl, aryl, halogen, hydroxyl,
hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy,
aryloxy, carboxamide, alkyl ester, or aryl ester; wherein preferably
alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy, and/or alkyl
ester contains lower alkyl, straight or branched, more preferably having
1-6 C-atoms. 180. The intermediate product of any one of embodiments
166-179, wherein R1 and R2 independently represent hydrogen, lower alkyl,
or lower alkoxy, wherein the lower alkyl and lower alkoxy, independently,
have 1-5 C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms.
181. The intermediate product of any one of embodiments 166-180, wherein
R1 and R2 independently represent alkyl having 1-2 C-atoms (ethyl, or
methyl), or alkoxy having 1-2 C-atoms (ethoxy, or methoxy). 182. The
intermediate product of any one of embodiments 166-181, wherein R1 and R2
are methyl or methoxy, preferably methyl. 183. The intermediate product
of any one of embodiments 166-182, wherein R1 and R2 are both methyl.
184. The intermediate product of any one of embodiments 166-183, wherein
PG2 is OH. 185. An intermediate product of formula Chem. 50 or Chem.
51: wherein Q represents a bond, or a group of formula Chem. 6:

--(C(R15)(R16))q--, Chem. 6

wherein q is 1-6, and R15 and R16 independently of each other and
independently for each value of q represent hydrogen, alkyl, carboxyl, or
hydroxyl; R represents hydrogen, or alkyl; R1 and R2 independently
represent (i) alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl,
hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy,
aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester,
alkyl sulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together form cyclo
alkyl, heterocyclyl, or heteroaryl; and each of PG1 and PG2
represents a protection group, preferably as defined in any one of
embodiments 167-173; or a pharmaceutically acceptable salt, amide, or
ester thereof. 186. An intermediate compound selected from Chem. 23,
Chem. 24, Chem. 25, Chem. 26, Chem. 27, Chem. 28, and Chem. 29; or a
pharmaceutically acceptable salt, amide, or ester thereof. 187. The
compound of Chem. 40; or a pharmaceutically acceptable salt, amide, or
ester thereof. 188. A compound characterised by the name of the compound
of Example 11 herein; or a pharmaceutically acceptable salt, amide, or
ester thereof. 189. The compound of embodiment 188, which is a compound
of embodiment 187. 190. A peptide intermediate product, which is selected
from the following analogues of GLP-1(9-37) (SEQ ID NO: 1): (i) (18K,
22E, 34Q); (ii) (30E, 36K, 38E); (iii) (31H, 34Q); (iv) 34R; (v) (34R,
37K); and (vi) (34R, 37K, 38E); or a pharmaceutically acceptable salt,
amide, or ester thereof. 191. A peptide intermediate product, which is
the following analogue of GLP-1A(3-37) (SEQ ID NO: 3): (17R, 20R, 33R,
38K); or a pharmaceutically acceptable salt, amide, or ester thereof.

EXAMPLES

[0245] This experimental part starts with a list of abbreviations, and is
followed by a section including general methods for synthesising and
characterising peptides and derivatives of the invention. Then follows a
number of examples which relate to the preparation of specific GLP-1
peptide derivatives, and at the end a number of examples have been
included relating to the activity and properties of these peptides and
derivatives (section headed pharmacological methods).

[0319] This section relates to methods for solid phase peptide synthesis
(SPPS methods, including methods for de-protection of amino acids,
methods for cleaving the peptide from the resin, and for its
purification), as well as methods for detecting and characterising the
resulting peptide (LCMS, MALDI, and UPLC methods). The solid phase
synthesis of peptides may in some cases be improved by the use of
di-peptides protected on the di-peptide amide bond with a group that can
be cleaved under acidic conditions such as, but not limited to,
2-Fmoc-oxy-4-methoxybenzyl, or 2,4,6-trimethoxybenzyl. In cases where a
serine or a threonine is present in the peptide, pseudoproline
di-peptides may be used (available from, e.g., Novabiochem, see also W.
R. Sampson (1999), J. Pep. Sci. 5, 403). The protected amino acid
derivatives used were standard Fmoc-amino acids (supplied from e.g.
Anaspec, IRIS, or Novabiochem). The N-terminal amino acid was Boc
protected at the alpha amino group (e.g. Boc-His(Boc)-OH, or
Boc-His(Trt)-OH for peptides with H is at the N-terminus). The epsilon
amino group of lysines in the sequence were either protected with Mtt,
Mmt, Dde, ivDde, or Boc, depending on the route for attachment of the
albumin binding moiety and spacer. The albumin binding moiety and/or
linker can be attached to the peptide either by acylation of the resin
bound peptide or by acylation in solution of the unprotected peptide. In
case of attachment of the albumin binding moiety and/or linker to the
protected peptidyl resin, the attachment can be modular using SPPS and
suitably protected building blocks such as but not limited to Fmoc-OEG-OH
(Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-Trx-OH (Fmoc-tranexamic
acid), Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,
nonadecanedioic acid mono-tert-butyl ester, or
4-(9-carboxynonyloxy)benzoic acid tert-butyl ester.

1. Synthesis of Resin Bound Peptide

SPPS Method A

[0320] SPPS method A refers to the synthesis of a protected peptidyl resin
using Fmoc chemistry on an Applied Biosystems 433 peptide synthesiser
(also designated ABI433A synthesiser) in 0.25 mmol or 1.0 mmol scale
using the manufacturer's FastMoc UV protocols which employ HBTU or HATU
mediated couplings in NMP, and UV monitoring of the de-protection of the
Fmoc protection group.

[0321] The starting resin used for the synthesis of peptide amides was a
suitable Rink-Amide resin (for peptide amides), or (for peptides with a
carboxy C-terminus) either a suitable Wang resin or a suitable
chlorotrityl resin. Suitable resins are commercially available from,
e.g., Novabiochem.

SPPS Method B

[0322] SPPS method B refers to the synthesis of a protected peptidyl resin
using Fmoc chemistry on a microwave-based Liberty peptide synthesiser
(CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load
Wang resin available from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang
resin, 0.35 mmol/g). Fmoc-deprotection was with 5% piperidine in NMP at
up to 70 or 75° C. The coupling chemistry was DIC/HOAt in NMP.
Amino acid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold)
were added to the resin followed by the same molar equivalent of DIC
(0.75M in NMP). For example, the following amounts of 0.3M amino
acid/HOAt solution were used per coupling for the following scale
reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml.
Coupling times and temperatures were generally 5 minutes at up to 70 or
75° C. Longer coupling times were used for larger scale reactions,
for example 10 min. Histidine amino acids were double coupled at
50° C., or quadruple coupled if the previous amino acid was
sterically hindered (e.g. Aib). Arginine amino acids were coupled at RT
for 25 min then heated to 70 or 75° C. for 5 min. Some amino acids
such as but not limited to Aib, were "double coupled", meaning that after
the first coupling (e.g. 5 min at 75° C.), the resin is drained
and more reagents are added (amino acid, HOAt and DIC), and the mixture
in heated again (e.g. 5 min at 75° C.). When a chemical
modification of a lysine side chain was desired, the lysine was
incorporated as Lys(Mtt). The Mtt group was removed by washing the resin
with DCM and suspending the resin in neat (undiluted)
hexafluoroisopropanol for 20 minutes followed by washing with DCM and
NMP. The chemical modification of the lysine was performed either by
manual synthesis (see SPPS method D) or by one or more automated steps on
the Liberty peptide synthesiser as described above, using suitably
protected building blocks (see General methods), optionally including a
manual coupling.

SPPS Method D

[0323] SPPS method D refers to synthesis of the protected peptidyl resin
using manual Fmoc chemistry. This was typically used for the attachment
of the linkers and side chains to the peptide backbone. The following
conditions were employed at 0.25 mmol synthesis scale. The coupling
chemistry was DIC/HOAt/collidine in NMP at a 4-10 fold molar excess.
Coupling conditions were 1-6 h at room temperature. Fmoc-deprotection was
performed with 20-25% piperidine in NMP (3×20 ml, each 10 min)
followed by NMP washings (4×20 mL). Dde- or ivDde-deprotection was
performed with 2% hydrazine in NMP (2×20 ml, each 10 min) followed
by NMP washings (4×20 ml). Mtt- or Mmt-deprotection was performed
with 2% TFA and 2-3% TIS in DCM (5×20 ml, each 10 min) followed by
DCM (2×20 ml), 10% MeOH and 5% DIPEA in DCM (2×20 ml) and NMP
(4×20 ml) washings, or by treatment with neat hexafluoroisopropanol
(5×20 ml, each 10 min) followed by washings as above. The albumin
binding moiety and/or linker can be attached to the peptide either by
acylation of the resin bound peptide or acylation in solution of the
unprotected peptide (see the routes described below). In case of
attachment of the albumin binding moiety and/or linker to the protected
peptidyl resin the attachment can be modular using SPPS and suitably
protected building blocks (see General methods).

[0327] The albumin binding moiety was dissolved in NMP/DCM (1:1, 10 ml).
The activating reagent such as HOBt (4 molar equivalents relative to
resin) and DIC (4 molar equivalents relative to resin) was added and the
solution was stirred for 15 min. The solution was added to the resin and
DIPEA (4 molar equivalents relative to resin) was added. The resin was
shaken 2 to 24 hours at room temperature. The resin was washed with NMP
(2×20 ml), NMP/DCM (1:1, 2×20 ml) and DCM (2×20 ml).

[0328] Attachment to Peptide in Solution--Route III:

[0329] Activated (active ester or symmetric anhydride) albumin binding
moiety or linker such as octadecanedioic acid
mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600) 1-1.5
molar equivalents relative to the peptide was dissolved in an organic
solvent such as acetonitrile, THF, DMF, DMSO or in a mixture of
water/organic solvent (1-2 ml) and added to a solution of the peptide in
water (10-20 ml) together with 10 molar equivalents of DIPEA. In case of
protecting groups on the albumin binding residue such as tert-butyl, the
reaction mixture was lyophilised overnight and the isolated crude peptide
deprotected afterwards. In case of tert-butyl protection groups the
deprotection was performed by dissolving the peptide in a mixture of
trifluoroacetic acid, water and triisopropylsilane (90:5:5). After for 30
min the mixture was evaporated in vacuo and the crude peptide purified by
preparative HPLC as described later.

SPPS Method E

[0330] SPPS method E refers to peptide synthesis by Fmoc chemistry on a
Prelude Solid Phase Peptide Synthesiser from Protein Technologies
(Tucson, Ariz. 85714 U.S.A.). A suitable resin is a pre-loaded, low-load
Wang resin available from Novabiochem (e.g. low load fmoc-Lys(Mtt)-Wang
resin, 0.35 mmol/g). Fmoc-deprotection was with 25% piperidine in NMP for
2×10 min. The coupling chemistry was DIC/HOAt/collidine in NMP.
Amino acid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold)
were added to the resin followed by the same molar equivalent of DIC (3 M
in NMP) and collidine (3 M in NMP). For example, the following amounts of
0.3M amino acid/HOAt solution were used per coupling for the following
scale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml. Coupling
times were generally 60 minutes. Some amino acids including, but not
limited to arginine, Aib or histidine were "double coupled", meaning that
after the first coupling (e.g. 60 min), the resin is drained and more
reagents are added (amino acid, HOAt, DIC, and collidine), and the
mixture allowed to react gain (e.g. 60 min). Some amino acids and fatty
acid derivatives including but not limited to Fmoc-Oeg-OH, Fmoc-Trx-OH,
Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,
nonadecanedioic acid mono-tert-butyl ester, or
4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were coupled for
prolonged time, for example 6 hours. When a chemical modification of a
lysine side chain was desired, the lysine was incorporated as Lys(Mtt).
The Mtt group was removed by washing the resin with DCM and suspending
the resin in hexafluoroisopropanol/DCM (75:25) for 3×10 minutes
followed by washings with DCM, 20% piperidine and NMP. The chemical
modification of the lysine was performed either by manual synthesis (see
SPPS method D) or by one or more automated steps on the Prelude peptide
synthesiser as described above using suitably protected building blocks
(see General methods).

2. Cleavage of Peptide from the Resin and Purification

[0331] After synthesis the resin was washed with DCM, and the peptide was
cleaved from the resin by a 2-3 hour treatment with TFA/TIS/water
(95/2.5/2.5 or 92.5/5/2.5) followed by precipitation with diethylether.
The peptide was dissolved in a suitable solvent (such as, e.g., 30%
acetic acid) and purified by standard RP-HPLC on a C18, 5 μM column,
using acetonitrile/water/TFA. The fractions were analysed by a
combination of UPLC, MALDI and LCMS methods, and the appropriate
fractions were pooled and lyophilised.

3. Methods for Detection and Characterisation

LCMS Methods

LCMS Method 1 (LCMS1)

[0332] An Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was
used to identify the mass of the sample after elution from an Agilent
1200 series HPLC system. The de-convolution of the protein spectra was
calculated with Agilent's protein confirmation software.

[0351] The following linear gradient was used: 65% A, 35% B to 25% A, 65%
B over 16 minutes at a flow-rate of 0.40 ml/min.

Method 08 B2 1

[0352] UPLC (method 08_B2--1): The RP-analysis was performed using a
Waters UPLC system fitted with a dual band detector. UV detections at 214
nm and 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130
Å, 1.7 um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H2O, 0.05% TFA

B: 99.95% CH3CN, 0.05% TFA

[0353] The following linear gradient was used: 95% A, 5% B to 40% A, 60% B
over 16 minutes at a flow-rate of 0.40 ml/min.

Method 08 B4 1

[0354] UPLC (method 08_B4--1): The RP-analysis was performed using a
Waters UPLC system fitted with a dual band detector. UV detections at 214
nm and 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130
Å, 1.7 um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H2O, 0.05% TFA

B: 99.95% CH3CN, 0.05% TFA

[0355] The following linear gradient was used: 95% A, 5% B to 95% A, 5% B
over 16 minutes at a flow-rate of 0.40 ml/min.

Method 05 B10 1

[0356] UPLC (Method 05_B10--1): The RP-analyses was performed using a
Waters UPLC system fitted with a dual band detector. UV detections at 214
nm and 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130
Å, 1.7 um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na2SO4, 0.04 M H3PO4, 10% CH3CN (pH 3.5)

B: 70% CH3CN, 30% H2O

[0357] The following linear gradient was used: 40% A, 60% B to 20% A, 80%
B over 8 minutes at a flow-rate of 0.40 ml/min.

Method 02 B4 4

[0358] UPLC (Method 02_B4--4): The RP-analysis was performed using a
Alliance Waters 2695 system fitted with a Waters 2487 dualband detector.
UV detections at 214 nm and 254 nm were collected using a Symmetry300
C18, 5 um, 3.9 mm×150 mm column, 42° C. Eluted with a linear
gradient of 5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid
(1.0%) in water over 15 minutes at a flow-rate of 1.0 ml/min.

Method 01 B4 1

[0359] HPLC (Method 01_B4--1): The RP-analysis was performed using a
Waters 600S system fitted with a Waters 996 diode array detector. UV
detections were collected using a Waters 3 mm×150 mm 3.5 um C-18
Symmetry column. The column was heated to 42° C. and eluted with a
linear gradient of 5-95% acetonitrile, 90-0% water, and 5%
trifluoroacetic acid (1.0%) in water over 15 minutes at a flow-rate of 1
ml/min.

MALDI-MS Method

[0360] Molecular weights were determined using matrix-assisted laser
desorption and ionisation time-of-flight mass spectroscopy, recorded on a
Microflex or Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy
cinnamic acid was used.

[0362] Overnight reflux of the C12, C14, C16 and C18 diacids with
Boc-anhydride, DMAP, and t-butanol in toluene gives predominately the
t-butyl mono ester. Obtained is after work-up a mixture of mono acid,
diacid and diester. Purification is carried out by washing, short plug
silica filtration and crystallisation.

2. Synthesis of 2-(1-Trityl-1H-imidazol-4-yl)-ethyl amine

##STR00023##

[0364] Histamine dihydrochloride (20.47 g; 0.111 mol) and triethylamine
(48 mL; 0.345 mol) in absolute methanol (400 mL) were stirred at room
temperature for 10 min. Trifluoroacetic acid ethyl ester (14.6 mL; 0.122
mol) in methanol (30 mL) was added dropwise over 30 min at 0° C.
Reaction mixture was stirred for 3.5 hrs at room temperature and then it
was evaporated to dryness in vacuo. The residue was dissolved in
dichlormethane (450 mL) and triethylamine (31 mL; 0.222 mol) was added.
Then trityl chloride (34.1 g; 0.122 mol) was added piecewise and mixture
was stirred over night at room temperature. Chloroform (400 mL) and water
(600 mL) were poured into reaction mixture. Aqueous layer was separated
and extracted with chloroform (3×400 mL). The combined organic
layers were dried over anhydrous magnesium sulfate. Solvent was removed
and the beige solid was triturated with hexanes (1000 mL). Suspension was
filtered to yield
2,2,2-trifluoro-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-acetamide as
white solid.

[0367] The above amide (45.54 g; 0.101 mmol) was dissolved in
tetrahydrofuran (1000 mL) and methanol (1200 mL). A solution of sodium
hydroxide (20.26 g; 0.507 mol) in water (500 mL) was added. Mixture was
stirred for 2 hrs at room temperature and then it was concentrated in
vacuo. The residue was separated between chloroform (1200 mL) and water
(800 mL). Aqueous layer was extracted with chloroform (3×400 mL).
Organic layers were combined and dried over anhydrous magnesium sulfate.
Evaporation of the solvent yielded brown oil, which was dried for 3 days
in vacuo to give the title product as beige solid.

[0377] A solution of 2-(1-Trityl-1H-imidazol-4-yl)-ethyl amine (5.00 g,
14.2 mmol) prepared as described above and triethylamine (9.86 mL, 70.7
mmol) in toluene (80 mL) was added dropwise over 50 min to a solution of
the above dione compound (3.65 g, 21.2 mmol) in toluene (40 mL) at
75° C. The mixture was stirred at this temperature for additional
3 hrs (until the starting amine was detected on TLC), then it was
evaporated to dryness. The residue was redissolved in chloroform (300 mL)
and washed with 10% aqueous solution of citric acid (200 mL). The aqueous
phase was extracted with chloroform (2×60 mL); the chloroform
phases were combined, dried over anhydrous magnesium sulfate and solvent
removed in vacuo. The residue was triturated with hot chloroform (140
mL); hexanes (70 mL) were added and the suspension was stirred at room
temperature overnight. Solids were filtered off, washed with
chloroform/hexanes mixture (1:1, 2×50 mL) and dried in vacuo to
give the title product.

[0383] Aluminum chloride powder (80.0 g, 600 mmol) was added in portions
to a stirred mixture of tert-butylbenzene (40.0 g, 300 mmol) and succinic
anhydride (26.7 g, 267 mmol) and 1,1,2,2-tetrachloroethane (100 mL).
After all the aluminum chloride had been added, the mixture was poured
into a mixture of ice (500 mL) and concentrated hydrochloric acid (100
mL). The organic layer was separated, washed with water (500 mL) and the
solvent distilled off. Solid residue was dissolved in hot 15% aqueous
solution of sodium carbonate (1000 mL), filtered, cooled and the acid was
precipitated with hydrochloric acid (acidified to pH=1). The crude acid
was filtered, dried on air and recrystalised from benzene (500 mL) to
give 4-(4-tert-butyl-phenyl)-4-oxo-butyric acid as colorless crystals.

[0387] A mixture of the above acid (36.0 g, 154 mmol), potassium hydroxide
(25.8 g, 462 mmol), hydrazine hydrate (20 mL, 400 mmol) and ethylene
glycol (135 mL) was refluxed for 3 hrs, and then distilled until the
temperature of the vapor had risen to 196-198° C. After a further
14 hrs reflux, the mixture was allowed to cool slightly, and was then
poured into cold water (200 mL). The mixture was acidified with
concentrated hydrochloric acid (to pH=1) and extracted with
dichloromethane (2×400 mL). The organic extracts were combined,
dried over anhydrous magnesium sulfate, solvent removed in vacuo and the
residue was purified by column chromatography (Silicagel 60A, 0.060-0.200
mm; eluent: hexanes/ethyl acetate 10:1-6:1) to give the title product as
off white solid.

[0393] Hydroxylamine hydrochloride (15.9 g, 229 mmol) was added to a
solution of 4(5)-imidazolecarboxaldehyde (20.0 g, 209 mmol) and sodium
carbonate (12.1 g, 114 mmol) in water (400 mL) and the resulting solution
was stirred at room temperature overnight. The mixture was evaporated to
100 mL and cooled in an ice bath. The solids were separated by filtration
and the filtrate was concentrated to 40 mL. After cooling to 0°
C., another portion of crystals was obtained. The solids (23 g) were
combined and recrystallised from ethanol (approx. 160 mL) to afford
imidazole-4(5)-carbaldehyde oxime as colorless crystals.

[0396] Acetyl chloride (51.0 mL, 718 mmol) was added dropwise to methanol
(670 mL) at 0° C. under argon. After 30 min, the cooling bath was
removed and the above oxime (16.0 g, 144 mmol) was added, followed by
palladium on carbon (5 wt %, 6.1 g). The mixture was hydrogenated at
atmospheric pressure for 17 hrs, then it was filtered through Celite and
the solvent evaporated to give pure 4-(aminomethyl)-imidazole
dihydrochloride as colorless crystals.

[0403] The above amide (46.6 g; 107 mmol) was dissolved in tetrahydrofuran
(600 mL) and ethanol (310 mL). A solution of sodium hydroxide (21.4 g;
535 mmol) in water (85 mL) was added. Mixture was stirred for 5 hrs at
room temperature and then it was concentrated in vacuo. The residue was
separated between chloroform (1600 mL) and water (800 mL). Aqueous layer
was extracted with chloroform (4×200 mL). Organic layers were
combined and dried over anhydrous magnesium sulfate. Evaporation of the
solvent yielded (1-trityl-1H-imidazol-4-yl)-methylamine as off white
solid.

[0406] A solution of the above amine (10.0 g, 29.5 mmol) and triethylamine
(20.5 mL, 147 mmol) in toluene (220 mL) was added dropwise over 45 min to
a solution of 2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione (3.65 g, 21.2
mmol) in toluene (80 mL) at 75° C. The mixture was stirred at this
temperature for additional 3 hrs (until the starting amine was detected
on TLC), then it was evaporated to dryness. The residue was redissolved
in chloroform (500 mL) and washed with 10% aqueous solution of citric
acid (300 mL). The aqueous phase was extracted with chloroform (100 mL);
the chloroform phases were combined, washed with water (150 mL) dried
over anhydrous magnesium sulfate and solvent removed in vacuo. The
residue was purified by flash column chromatography (silica gel Fluke 60,
dichloromethane/methanol 98:2 to 9:1) and crystallised from
chloroform/hexanes mixture to give the title product as beige crystals.

[0416] Methanesulfonyl chloride (8 mL, 104 mmol) was added dropwise to a
solution of the above alcohol (32.0 g, 86.8 mmol) in dichloromethane (400
mL) and triethyl amine (15.5 mL) at 0° C. during 1 hr. The mixture
was stirred without cooling for an additional 1 hr; then it was washed
with 5% sodium bicarbonate and dried over anhydrous magnesium sulfate.
Dichloromethane was evaporated at 30° C. in vacuo and the residual
oily mesylate was used directly in the next step.

[0423] The above imidazole derivative (26.6 g, 53.5 mmol) was dissolved in
ethanol (300 mL) and tetrahydrofuran (150 mL) at 60° C., hydrazine
hydrate (50 g, 1 mol) was added and the solution was refluxed for 6 hrs
and then heated at 70° C. overnight. The solid was removed by
filtration and the filtrate was treated with 25% aqueous solution of
ammonia (2.5 l) and dichloromethane (2.5 L). The organic layer was dried
over anhydrous potassium carbonate and evaporated to give a residue,
which was purified by column chromatography on silica gel (Fluke 60,
chloroform saturated with ammonia/methanol) giving the title compound as
white solid.

[0480] Preparation method: SSPS method B.
2,2-Dimethyl-N-pyridin-2-ylmethyl-malonamic acid was coupled using the
same coupling condition as used for
2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid in
the previous examples. Fmoc-Glu-OtBu and 4-(9-carboxy-nonyloxy)-benzoic
acid tert-butyl ester (prepared as described in Example 25, step 2 of WO
2006/082204) were coupled using SPPS method D

[0481] UPLC (method 08_B4--1): Rt=8.98 min

[0482] LCMS4: Rt=2.23 min. m/z=1624(m/3), 1218 (m/4)

Pharmacological Methods

Example 13

In Vitro Potency

[0483] The purpose of this example is to test the activity, or potency, of
the GLP-1 receptor agonist derivatives in vitro.

[0484] The potencies of the GLP-1 receptor agonist derivatives of Examples
1-12 were determined as described below, i.e. as the stimulation of the
formation of cyclic AMP (cAMP) in a medium containing membranes
expressing the human GLP-1 receptor.

[0485] It is noted that the compound of Example 11 (Chem. 40) is a
comparative compound based on compound 215 (p. 24) of WO 2004/067548,
which according to FIG. 1 of this WO publication is one of the most
potent compounds of this publication

[0486] Principle

[0487] Purified plasma membranes from a stable transfected cell line,
BHK467-12A (tk-ts13), expressing the human GLP-1 receptor were stimulated
with the GLP-1 receptor agonist derivative in question, and the potency
of cAMP production was measured using the AlphaScreen® cAMP Assay Kit
from Perkin Elmer Life Sciences. The basic principle of The AlphaScreen
Assay is a competition between endogenous cAMP and exogenously added
biotin-cAMP. The capture of cAMP is achieved by using a specific antibody
conjugated to acceptor beads.

[0488] Cell Culture and Preparation of Membranes

[0489] A stable transfected cell line and a high expressing clone were
selected for screening. The cells were grown at 5% CO2 in DMEM, 5%
FCS, 1% Pen/Strep (Penicillin/Streptomycin) and 0.5 mg/ml of the
selection marker G418.

[0490] Cells at approximate 80% confluence were washed 2× with PBS
and harvested with Versene (aqueous solution of the tetrasodium salt of
ethylenediaminetetraacetic acid), centrifuged 5 min at 1000 rpm and the
supernatant removed. The additional steps were all made on ice. The cell
pellet was homogenised by the Ultrathurax for 20-30 sec. in 10 ml of
Buffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at
20.000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mM
Na-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenised for 20-30
sec and centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2,
homogenisation and centrifugation was repeated once and the membranes
were resuspended in Buffer 2. The protein concentration was determined
and the membranes stored at -80° C. until use.

[0491] The assay was performed in 1/2-area 96-well plates, flat bottom
(Costar cat. no: 3693). The final volume per well was 50 μl.

[0496] Suitable dilution series in AlphaScreen Buffer were prepared of the
cAMP standard as well as the GLP-1 analogue or derivative to be tested,
e.g. the following eight concentrations of the GLP-1 compound: 10-7,
10-8, 10-9, 10-10, 10-11, 10-12, 10-13 and
10-14M, and a series from, e.g., 10-6 to 3×10-11 of
cAMP.

[0497] Membrane/Acceptor Beads

[0498] Use hGLP-1/BHK 467-12A membranes; 6 μg/well corresponding to 0.6
mg/ml (the amount of membranes used pr. well may vary)

[0505] As the beads are sensitive to direct light, any handling was in the
dark (as dark as possible), or in green light. All dilutions were made on
ice.

[0506] Procedure

1. Make the AlphaScreen Buffer.

[0507] 2. Dissolve and dilute the GLP-1/Analogues/cAMP standard in
AlphaScreen Buffer. 3. Make the Donor Beads solution and incubate 30 min.
at R.T. 4. Add the cAMP/GLP-1/Analogues to the plate: 10 μl per well.
5. Prepare membrane/Acceptor Beads solution and add this to the plates:
10 μl per well. 6. Add the Donor Beads: 30 μl per well. 7. Wrap the
plate in aluminum foil and incubate on the shaker for 3 hours (very
slowly) at RT. 8. Count on AlphaScreen--each plate pre incubates in the
AlphaScreen for 3 minutes before counting.

[0508] If desired, the fold variation in relation to GLP-1 may be
calculated as EC50 (GLP-1)/EC50 (analogue)--3693.2.

[0511] All tested derivatives of the invention had a good in vitro potency
corresponding to an EC50 of 2100 pM or below; eight derivatives were
even more potent having and EC50 at 1000 pM or below; five
derivatives had a still further improved potency corresponding to an
EC50 at 500 pM or below; four derivatives were very potent
corresponding to an EC50 at 300 pM or below; and one derivative had
a very good potency corresponding to an EC50 at 100 pM or below.

[0512] The comparative compound of Example 11 was much less potent, namely
with an EC50 of above 8000 pM.

Example 14

GLP-1 Receptor Binding

[0513] The purpose of this experiment is to investigate the binding to the
GLP-1 receptor of the GLP-1 agonist derivatives, and how the binding is
potentially influenced by the presence of albumin. This is done in an in
vitro experiment as described below.

[0514] The binding affinity of the GLP-1 receptor agonist derivatives of
Examples 1-12 to the human GLP-1 receptor was measured by way of their
ability to displace of 125I-GLP-1 from the receptor.

[0515] It is noted that the compound of Example 11 (Chem. 40) is a
comparative compound based on compound 215 (p. 24) of WO 2004/067548,
which according to FIG. 1 of this WO publication is one of the most
potent compounds of this publication.

[0516] In order to test the binding of the derivatives to albumin, the
assay was performed with a low concentration of albumin
(0.005%--corresponding to the residual amount thereof in the tracer), as
well as with a high concentration of albumin (2.0% added).

[0517] A shift in the binding affinity, IC50, is an indication that
the peptide in question binds to albumin, and thereby a prediction of a
potential protracted pharmacokinetic profile of the peptide in question
in animal models.

Conditions

[0518] Species (in vitro): Hamster

[0519] Biological End Point: Receptor Binding

[0520] Assay Method: SPA

[0521] Receptor: GLP-1 receptor

[0522] Cell Line: BHK tk-ts13

Cell Culture and Membrane Purification

[0523] A stable transfected cell line and a high expressing clone were
selected for screening. The cells were grown at 5% CO2 in DMEM, 10%
FCS, 1% Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the
selection marker G418.

[0524] The cells (approx. 80% confluence) were washed twice in PBS and
harvested with Versene (aqueous solution of the tetrasodium salt of
ethylenediaminetetraacetic acid), following which they were separated by
centrifugation at 1000 rpm for 5 min. The cells/cell pellet must be kept
on ice to the extent possible in the subsequent steps. The cell pellet
was homogenised with Ultrathurrax for 20-30 seconds in a suitable amount
of Buffer 1 (depending on the amount of cells, but e.g. 10 ml). The
homogenate was centrifuged at 20000 rpm for 15 minutes. The pellet was
resuspended (homogenised) in 10 ml Buffer 2 and re-centrifuged. This step
was repeated once more. The resulting pellet was resuspended in Buffer 2,
and the protein concentration was determined. The membranes were stored
at minus 80° C.

[0535] The IC50 value was read from the curve as the concentration
which displaces 50% of 125I-GLP-1 from the receptor, and the ratio
of [(IC50/nM) high HSA]/[(IC50/nM) low HSA] was determined.

[0536] Generally, the binding to the GLP-1 receptor at low albumin
concentration should be as good as possible, corresponding to a low
IC50 value.

[0537] The IC50 value at high albumin concentration is a measure of
the influence of albumin on the binding of the derivative to the GLP-1
receptor. As is known, the GLP-1 receptor agonist derivatives also bind
to albumin. This is a generally desirable effect, which extends their
lifetime in plasma. Therefore, the IC50 value at high albumin will
generally be higher than the IC50 value at low albumin,
corresponding to a reduced binding to the GLP-1 receptor, caused by
albumin binding competing with the binding to the GLP-1 receptor.

[0538] A high ratio (IC50 value (high albumin)/IC50 value (low
albumin)) may therefore be taken as an indication that the derivative in
question binds well to albumin (may have a long half-life), and also per
se binds well to the GLP-1 receptor (the IC50 value (high albumin)
is high, and the IC50 value (low albumin) is low).

Results

[0539] The following results were obtained, where "ratio" refers to
[(IC50/nM) high HSA]/[(IC50/nM) low HSA]):

[0540] All derivatives had a ratio above 10; ten were above 20; six
derivatives were above 30; four derivatives were above 50; and one
derivative was above 100. The comparative compound of Example 11, had a
ratio above 300.

[0541] Furthermore as regards IC50 (low albumin), all derivatives,
except the comparative compound of Example 11, had an IC50 (low
albumin) below 40 nM; all but one below 20 nM; all but four were below
10.0 nM; five were below 5.00 nM; and three derivatives were below 1.00
nM.

[0542] Finally as regards IC50 (high albumin), all derivatives of the
invention had an IC50 (high albumin) below 900.00 nM; nine were
below 500.00 nM; four were below 100.00 nM; and two derivatives were
below 50.00 nM. The IC50 (high albumin) for the comparative compound
of Example 11 was above 800.00 nM.

Example 15

Estimate of Oral Bioavailability

[0543] The purpose of this experiment is to estimate the oral
bioavailability of the GLP-1 receptor agonist derivatives.

[0544] To this end, the exposure in plasma after direct injection into the
intestinal lumen of the compounds is studied in vivo in rats, as
described in the following.

[0545] The compounds are tested in a concentration of 1000 uM in a
solution of 55 mg/ml sodium caprate.

[0546] 32 male Sprague Dawley rats with a body weight upon arrival of
approximately 240 g are obtained from Taconic (Denmark) and assigned to
the different treatments by simple randomisation, 4 rats per group. The
rats are fasted for approximately 18 hours before the experiment and
taken into general anaesthesia (Hypnorm/Dormicum).

[0547] The compounds are administered in the jejunum either in the
proximal part (10 cm distal for the duodenum) or in the mid-intestine (50
cm proximal for the cecum). A PE50-catheter, 10 cm long is inserted into
the jejunum, forwarded at least 1.5 cm into the jejunum, and secured
before dosing by ligature around the gut and the catheter with 3/0 suture
distal to tip to prevent leak or catheter displacement. Catheter is
placed without syringe and needle and 2 ml saline is administered into
abdomen before closing the incision with wound clips.

[0548] 100 μl of the respective compound is injected into the jejunal
lumen through the catheter with a 1 ml syringe. Subsequently, 200 μl
of air is pushed into the jejunal lumen with another syringe to "flush"
the catheter. This syringe is leaved connected to the catheter to prevent
flow back into the catheter.

[0549] Blood samples (200 ul) are collected at desired intervals (usually
at times 0, 10, 30, 60, 120 and 240 min) into EDTA tubes from the tail
vein and centrifuged 5 minutes, 10000G, at 4° C. within 20
minutes. Plasma (75 ul) is separated to Micronic tubes, immediately
frozen, and kept at -20° C. until analyzed for plasma
concentration of the respective GLP-1 receptor agonist derivative with
LOCI (Luminescent Oxygen Channeling Immunoassay), generally as described
for the determination of insulin by Poulsen and Jensen in Journal of
Biomolecular Screening 2007, vol. 12, p. 240-247. The donor beads are
coated with streptavidin, while acceptor beads are conjugated with a
monoclonal antibody recognising a mid-/C-terminal epitope of the peptide.
Another monoclonal antibody, specific for the N-terminus, is
biotinylated. The three reactants are combined with the analyte and
formed a two-sited immuno-complex. Illumination of the complex released
singlet oxygen atoms from the donor beads, which are channeled into the
acceptor beads and triggered chemiluminescence which is measured in an
Envision plate reader. The amount of light is proportional to the
concentration of the compound.

[0550] After the blood sampling the rats are sacrificed under anaesthesia
and the abdomen is opened to verify correct catheter placement.

[0551] The mean (n=4) plasma concentrations (pmol/l) are determined as a
function of time. The ratio of plasma concentration (pmol/l) divided by
the concentration of the dosing solution (μmol/l) is calculated for
each treatment, and the results for t=30 min (30 minutes after the
injection of the compound in the jejunum) are assessed (dose-corrected
exposure at 30 min) as a surrogate measure of intestinal bioavailability.
The dose-corrected exposure has been shown to correlate significantly
with the actual bioavailability.

[0552] The results may be given as dose-corrected exposure at 30 min which
refers to (the plasma concentration 30 minutes after injection of the
compound in the jejunum (pM)), divided by (the concentration of the
compound in the dosing solution (pM)).

Example 16

Pharmacokinetics in Minipigs

[0553] The purpose of this study is to determine the protraction in vivo
of the GLP-1 receptor agonist derivatives after i.v. administration to
minipigs, i.e. the prolongation of their time of action. This is done in
a pharmacokinetic (PK) study, where the terminal half-life of the
derivative in question is determined. By terminal half-life is generally
meant the period of time it takes to halve a certain plasma
concentration, measured after the initial distribution phase.

[0554] Male Gottingen minipigs obtained from Ellegaard Gottingen Minipigs
(Dalmose, Denmark) approximately 7-14 months of age and weighing from
approximately 16-35 kg are used in the studies. The minipigs are housed
individually and fed restrictedly once or twice daily with SDS minipig
diet (Special Diets Services, Essex, UK). After at least 2 weeks of
acclimatisation two permanent central venous catheters are implanted in
vena cava caudalis or cranialis in each animal. The animals are allowed 1
week recovery after the surgery, and are then used for repeated
pharmacokinetic studies with a suitable wash-out period between dosings.

[0555] The animals are fasted for approximately 18 h before dosing and for
at least 4 h after dosing, but had ad libitum access to water during the
whole period.

[0556] The compounds are dissolved in 50 mM sodium phosphate, 145 mM
sodium chloride, 0.05% tween 80, pH 7.4 to a concentration of usually
from 20-60 nmol/ml. Intravenous injections (the volume corresponding to
usually 1-2 nmol/kg, for example 0.033 ml/kg) of the compounds are given
through a catheter, and blood is sampled at predefined time points for up
till 13 days post dosing (preferably through the other catheter). Blood
samples (for example 0.8 ml) are collected in EDTA buffer (8 mM) and then
centrifuged at 4° C. and 1942G for 10 minutes. Plasma is pippetted
into Micronic tubes on dry ice, and kept at -20° C. until analyzed
for plasma concentration of the respective GLP-1 compound using ELISA or
a similar antibody based assay or LC-MS. Individual plasma
concentration-time profiles are analyzed by a non-compartmental model in
WinNonlin v. 5.0 (Pharsight Inc., Mountain View, Calif., USA), and the
resulting terminal half-lives (harmonic mean) determined.

[0557] While certain features of the invention have been illustrated and
described herein, many modifications, substitutions, changes, and
equivalents will now occur to those of ordinary skill in the art. It is,
therefore, to be understood that the appended claims are intended to
cover all such modifications and changes as fall within the true spirit
of the invention.